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Secondary metabolites from selected British Columbian marine organisms Tischler, Mark 1987

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SECONDARY METABOLITES  FROM SELECTED  BRITISH COLUMBIAN MARINE ORGANISMS  By  MARK TISCHLER  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Department o f Chemistry)  We a c c e p t t h i s t h e s i s  as conforming  to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987  ©  Mark T i s c h l e r ,  1987  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives.  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  /9^//  J?^  /9f?  - i i -  TABLE OF CONTENTS  Page ABSTRACT  iv  LIST OF TABLES  v  LIST OF FIGURES  vi  LIST OF SCHEMES  viii  LIST OF APPENDICES  x  ACKNOWLEDGEMENTS  ABBREVIATIONS  A.  xi  x i i  1  INTRODUCTION TO THE BRYOZOANS  1  i)  Biology  1  ii)  N a t u r a l P r o d u c t s Chemistry  3  B.  SYNTHESIS OF PHIDOLOPIN AND DESMETHYLPHIDOLOPIN  . . . .  16  C.  NITROPHENOLS FROM NORTHEAST PACIFIC BRYOZOANS  45  D.  DISCUSSION  54  E.  INTRODUCTION TO THE SPONGES  63  - iii -  F.  NOVEL STEROIDS FROM THE SPONGE A n t h r o a r c u a t a gracea  . . .  75  1.  Introduction  75  2.  I s o l a t i o n and S t r u c t u r e E l u c i d a t i o n  76  3.  A.  A4-3,6-diketosteroids  77  B.  A-Nor s t e r o l s  89  C.  Diosphenols  107  D.  B i o g e n e s i s o f Sponge M e t a b o l i t e s  115  G.  EXPERIMENTAL  120  H.  APPENDICES  139  I.  BIBLIOGRAPHY  140  iv -  ABSTRACT  The (37), order  two  p u r i n e a l k a l o i d s , p h i d o l o p i n (36) and d e s m e t h y l p h i d o l o p i n  o r i g i n a l l y i s o l a t e d from P h i d o l o p o r a p a c i f i c a . were s y n t h e s i z e d i n to  produce  sufficient  quantities  of  the n a t u r a l p r o d u c t s f o r  extended b i o l o g i c a l and p h a r m a c o l o g i c a l s c r e e n i n g p r e v i o u s s t r u c t u r a l assignment Various  combinations  and  to  confirm  the  o f 37 which was based on s p e c t r a l d a t a .  o f p h i d o l o p i n (36), d e s m e t h y l p h i d o l o p i n (37),  4-hydroxymethyl-2-nitrophenol  (38)  and  4-methoxymethyl-2-nitrophenol  (39) were i s o l a t e d from f o u r d i f f e r e n t s p e c i e s o f bryozoans, D i a p e r o e c i a californica.  Heteropora a l a s k e n s i s .  Hippodiplosia insculpta.  A  dietary  T r i c e l l a r i a ternata origin  is  suggested  and  for  these  metabolites. The r e d sponge, A n t h o a r c u a t a graceae. y i e l d e d including  the  ,6-diketosteroids  116,  117,  s i x novel  steroids  the  steroids  A-nor  a n t h o s t e r o n e A (118) and anthosterone B (119) as w e l l as two containing steroids,  120 and 121.  The proposed s t r u c t u r e s were based on  a combination o f s p e c t r a l a n a l y s i s , chemical sis,  diosphenol  and s i n g l e c r y s t a l X-ray d i f f r a c t i o n  interconversions,  analysis.  synthe-  -  V  -  LIST OF TABLES Table  Page  1  80 MHz  ^H NMR  d a t a on p r o t e c t e d n i t r o p h e n o l s  41 and 42 2  80 MHz  3  80 MHz  X  H ^  22 NMR NMR  data comparison o f 53 and 54 data f o r p h i d o l o p i n  and d e s m e t h y l p h i d o l o p i n  (36)  (37)  40  1 3  5  l  n  NMR  d a t a f o r i s o l a t e d n i t r o p h e n o l s 36,  37  . .  48  6  1  H  NMR  d a t a f o r i s o l a t e d n i t r o p h e n o l s 38,  39  . .  50  7  l  U  NMR  data f o r A -3,6-ketosteroids  8  1 3  9  1  C  NMR  30  4  10  C  . . .  NMR  d a t a comparison f o r 36 and 37  4  44  (CDCI3)  data f o r A -3,6-ketosteroids 4  ...  83  (CDCI3) . . .  86  H NMR d a t a f o r anthosterone A (118) and anthosterone B (119) C NMR d a t a f o r anthosterone A (118) and a n t h o s t e r o n e B (119)  94  1 3  95  11  •'-H NMR  comparison between 119  and 123  12  ^-H NMR  d a t a f o r d i o s p h e n o l s 120  13  N i t r o p h e n o l s from N o r t h e a s t P a c i f i c Bryozoans  and 121  101 . . . . . .  110 131  - vi-  LIST OF FIGURES  Figure  Page  1  80 MHz ^-H NMR  2  80 MHz H NMR  3  80 MHz *H NMR o f MOM p r o t e c t e d p h i d o l o p i n 55  4  1  o f compound 41  20  o f compound 42  80 MHz H NMR o f MOM p h i d o l o p i n 56 1  protected  21 desmethyl33 desmethyl34  5  80 MHz H NMR  o f p h i d o l o p i n 36  6  80 MHz H NMR  o f d e s m e t h y l p h i d o l o p i n 37  7  100 MHz  8  75 MHz  9  80 MHz H NMR  f o r 4 - h y d r o x y m e t h y l - 2 - n i t r o p h e n o l 38  51  10  80 MHz H NMR  f o r 4-methoxymethyl-2-nitrophenol 39  52  11  400 MHz *H NMR  12  400 MHz H NMR  o f compound 122  81  13  75 MHz  o f compound 122  82  14  400 MHz %  15  75 MHz  16  X  l  1 3  C  C  1 3  NMR  X  1  1  C  1 3  C  NMR NMR  39 42  . . . .  o f compound 116  43  80  o f compound 117  84  o f compound 117  85  400 MHz H NMR  o f a n t h o s t e r o n e A 118  90  17  75 MHz  NMR  o f anthosterone A 118  91  18  400 MHz H NMR  o f a n t h o s t e r o n e B 119  92  19  75 MHz  o f anthosterone B 119  93a  20  APT spectrum f o r anthosterone B 119  21  270 MHz ^-H NMR  1 3  NMR  . . . .  o f p h i d o l o p i n 36 o f d e s m e t h y l p h i d o l o p i n 37  NMR  38  1  1 3  C  1  1 3  C  NMR  o f anthosterone B - a c e t a t e 123  93b . .  100  - vii -  22  A l l y l i c methylene system  23  SINEPT experiment  24  2D Homonuclear COSY on a n t h o s t e r o n e B 119  25  Computer generated ORTEP drawing o f  on anthosterone B 119  102 . . . .  105  . . . .  106  a n t h o s t e r o n e A 118 26  400 MHz H NMR o f compound E 120  27  2D Homonuclear COSY spectrum o f compound F 121 . .  28  400 MHz H NMR o f compound F 121  X  1  106 108 112 113  - viii LIST OF SCHEMES  Scheme  1  Page  S y n t h e t i c p l a n f o r p h i d o l o p i n (36) and d e s m e t h y l p h i d o l o p i n (37) 40  17  2  P r o t e c t i o n of 4-methyl-2-nitrophenol  ....  3  I n t e r p r e t a t i o n o f t h e MS f r a g m e n t a t i o n o f 41  . .  23  4  I n t e r p r e t a t i o n o f t h e MS f r a g m e n t a t i o n o f 42  . .  23  5  A l k y l a t i o n o f t h e o p h y l l i n e (45) w i t h 47  6  A l k y l a t i o n o f t h e o p h y l l i n e w i t h 49  7  B r o m i n a t i o n o f p r o t e c t e d n i t r o p h e n o l 41  8  MS f r a g m e n t a t i o n o f compound 51  9  B r o m i n a t i o n o f MOM p r o t e c t e d n i t r o p h e n o l 42  ....  18  25 26  ....  26 27  . . .  10  A l k y l a t i o n o f t h e o p h y l l i n e (45) w i t h 51  ....  11  B r o m i n a t i o n and a l k y l a t i o n  12  I n t e r p r e t a t i o n o f t h e MS f r a g m e n t a t i o n o f 55  13  A l k y l a t i o n o f t h e o p h y l l i n e (45) under v a r i e d conditions  28 29 31  . .  35 36  14  D e p r o t e c t i o n o f 53 w i t h c h l o r o t r i m e t h y l s i l a n e  . .  15  D e p r o t e c t i o n o f 55 and 56 w i t h d i l u t e a c i d  16  MS f r a g m e n t a t i o n o f p h i d o l o p i n (36)  17  MS f r a g m e n t a t i o n o f d e s m e t h y l p h i d o l o p i n (37)  18  MS f r a g m e n t a t i o n o f n i t r o p h e n o l 38  53  19  MS f r a g m e n t a t i o n o f n i t r o p h e n o l 39  53  20  B i o s y n t h e s i s o f x e s t o s t e r o l (95)  71  ...  37 37 49  . .  49  - ix -  21  Biosynthetic conversion of cholesterol 3B-hydroxymethyl-A-nor-cholestane  into 73  22  Jones o x i d a t i o n o f c h o l e s t e r o l  23  MS f r a g m e n t a t i o n o f compound B 117  24  MS f r a g m e n t a t i o n o f anthosterone A (118)  . . . .  97  25  MS f r a g m e n t a t i o n o f anthosterone B (119)  . . . .  98  26  A c e t y l a t i o n o f anthosterone B (119)  27  MS f r a g m e n t a t i o n o f 120  109  28  MS f r a g m e n t a t i o n o f compound F 121  114  29  B i o s y n t h e t i c p r o p o s a l f o r compounds A and B  30  B i o s y n t h e t i c p r o p o s a l f o r A-nor s t e r o i d s 118,  (83)  119  79 87  99  . . .  116  118  31  Formation o f d i o s p h e n o l s 120, 121  119  32  B i o s y n t h e t i c proposal f o r shortened side chains  119  -  X  -  LIST OF APPENDICES  Appendix 1  B i o a s s a y r e s u l t s f o r p h i d o l o p i n (36) d e s m e t h y l p h i d o l o p i n (37)  and 139  xi -  ACKNOWLEDGEMENTS  I would l i k e t o express my a p p r e c i a t i o n Andersen  f o r h i s encouragement  to  and guidance  Professor  throughout  Raymond  t h e course o f  t h i s work, and f o r h i s a s s i s t a n c e d u r i n g the p r e p a r a t i o n o f t h i s A l s o , I w i s h t o thank a number o f people who have the c o l l e c t i o n o f the organisms  studied, especially  thank Drs. G.K. E i g e n d o r f and S.O. Chan and t h e i r  assisted  thesis. me  Mr. Mike LeBlanc. staff  J.  in I  for efficient  a s s i s t a n c e and c o o p e r a t i o n i n the c o l l e c t i o n o f s p e c t r o s c o p i c d a t a . Finally,  I  wish t o extend a v e r y s p e c i a l  thanks t o my p a r e n t s f o r  t h e i r p a t i e n c e , c o n s t a n t encouragement and support throughout o f my s t u d i e s .  the course  - xii -  ABBREVIATIONS  CDCI3  -  Chloroform-  CHCI3  =  Chloroform  DMSO  -  Dimethylsulfoxide  EtOAc  -  Ethyl acetate  g  -  Grease peak  HPLC  -  High performance l i q u i d  HRMS  -  High r e s o l u t i o n mass spectrum  IR  -  Infrared  MS  -  Low r e s o l u t i o n mass spectrum  Na2S04  -  Sodium s u l f a t e  ^•H NMR  -  P r o t o n n u c l e a r magnetic  -  Carbon-13 n u c l e a r magnetic  NOE  -  N u c l e a r Overhauser enhancement  mp  -  Melting point  RT  -  Room temperature  S  -  Solvent  SCUBA  -  S e l f c o n t a i n e d underwater b r e a t h i n g apparatus  TLC  -  Thin layer  U  -  Unknown i m p u r i t y  UV  -  Ultraviolet  NMR  chromatography  (anhydrous) resonance resonance  signal  chromatography signal  - xiii -  Abbreviations  f o r m u l t i p l i c i t i e s o f NMR  s  =  singlet  d  =  doublet  t  =  triplet  q  =  quartet  dd =  d o u b l e t or d o u b l e t s  bs =  broad  m  multiplet  -  signals:  singlet  T h i s t h e s i s has been w r i t t e n i n conformance w i t h the "Handbook Authors", D.C.,  1978.  published  for  by the American Chemical S o c i e t y ; Washington,  - 1 -  A.  Bryozoans,  INTRODUCTION TO  commonly  THE  BRYOZOANS  found i n abundance i n p r e d a t o r - r i c h , competi-  t i v e environments, have demonstrated the a b i l i t y to grow on a l l forms o f hard  surface  steel.  The  prevalent  ecological  space such as r o c k s ,  a d a p t a b i l i t y o f bryozoans has groups  of  f o u l i n g organisms.  made  shells, them  c o r a l s , wood  one  of  For example, some 130  have been taken from s h i p bottoms, where they show a g e n e r a l to  anti fouling paints.^  the  and most  species  resistance  T h e i r remarkable a d h e s i o n p r o p e r t i e s have l e d  to f u l l  s c a l e s t u d i e s on t h e i r s e c r e t o r y p r o d u c t s i n the hope o f f i n d i n g  viable  new  bioadhesive  materials  acceptable  f o r c l i n i c a l use by  the  d e n t a l and m e d i c a l p r o f e s s i o n s . ^ Of p a r t i c u l a r i n t e r e s t , however, are the chemical on marine bryozoans i n the p a s t  twenty y e a r s  which have y i e l d e d a number  o f n o v e l b i o l o g i c a l l y - a c t i v e secondary m e t a b o l i t e s . prompted  continuing  have as t h e i r g o a l s  chemical  the d i s c o v e r y o f new  and  answers r e g a r d i n g  I.  Biology  The  phylum  studies  (or  polyzoa  mately 4000 known l i v i n g s p e c i e s . classes,  Phylactolaemata,  The  These f i n d i n g s have  o f members o f t h i s phylum which biologically-active  the o r i g i n o f the i s o l a t e d  Bryozoa  s t u d i e s conducted  metabolites.  or E c t o p r o c t a ) phylum  Gymnolaemata, and  compounds  is  contains  divided  Stenolaemata.  approxi-  into The  three classes  - 2 Stenolaemata class  and Gymnolaemata i n c l u d e o n l y marine  Phylactolaemata  i s restricted  to  bryozoans  approximately  while  the  50 known f r e s h  water s p e c i e s . Bryozoans and  width  are c o l o n i a l f i l t e r  and  o c c u r i n a v a r i e t y o f m o r p h o l o g i c a l forms.  names used t o d e s c r i b e moss - a n i m a l s .  f e e d i n g animals which v a r y i n h e i g h t  these  forms  I n B r i t i s h Columbia  are  false-corals,  Some o f the  sea-mats,  and  a l l o f these forms a r e common.  examples i n c l u d e : H e t e r o p o r a p a c i f i c a  - coral like,  Bugula  sp.  -  Some moss-  animal type, and Membranipora membranacea - sea-mat type. Bryozoan z o o i d s which chitinous  colonies have  and  body  usually  s p e c i e s a r e encased  are b u i l t o f a r e p l i c a t e d s e r i e s o f i n d i v i d u a l walls about  which 0.5  are  mm  calcareous,  i n length.  gelatinous  I n d i v i d u a l s o f most  i n a n o n - l i v i n g envelopment t h a t c o n t a i n s an opening  (orifice)  f o r the p r o t r u s i o n  lophophore  used t o g a t h e r the s m a l l p l a n k t o n ( c h i e f l y diatoms  phytoplankton)  that  make  of  the  circular  up the bryozoans'  muscles  o r horse-shoe  diet.  body i s o c c u p i e d l a r g e l y by t h e s p a c i o u s coelom, a tract,  zooids  shaped  and  other  The i n t e r i o r o f the U  shaped  digestive  and t h e anus which opens a l o n g s i d e t h e lophophore.  z o o i d s o f a c o l o n y a r e a t t a c h e d through pores o r gaps i n the body Some  or  The wall.  i n a colony are modified f o r s p e c i a l i z e d f u n c t i o n s (poly-  morphism) such as f e e d i n g  (autozooid,  the  largest  zooid),  cleaning,  p r o t e c t i o n o r b r o o d i n g o f the young ( h e t e r o z o o i d s ) . Bryozoan  colonies  are hermaphroditic  z o o i d s o c c u r r i n g i n the same c o l o n y . egg  will  pass  I n most  with  b o t h male and female  species,  the  fertilized  i n t o a b r o o d i n g chamber p r o d u c i n g l a r v a e which a r e non-  f e e d i n g . The l a r v a e  have  a  very  brief  planktonic  existence  before  - 3 -  settling  on a h a r d s u r f a c e .  Non  b r o o d i n g bryozoans p o s s e s s l a r v a e w i t h  a f u n c t i o n a l d i g e s t i v e t r a c t which can larval  life  transform  Ii)  A f t e r settlement,  and  ecological  and  pharmacological  c a r r i e d out around 1960, showing  some  reported  the f i r s t  marine  larvae begins  aspects  of  bryozoans  bryozoans.  studies  of  3  to  Roughly 9-10%  In 1970,  tutes  murine  c a r c i n o s a r c o m a 256  P388  the U.S.  lymphocytic  t e s t l i n e s i n the r a t .  (PS  were  P e t t i t and  Day  extracts  of  in  National  leukemia  modern  coelenterates^  o f the organisms t e s t e d  l e v e l of a c t i v i t y against  been  l i t t l e work  first  and  evidence of a n t i n e o p l a s t i c a c t i v i t y 5  have  invertebrates  w i t h e x t r a c t s from s p o n g e s  a significant  P e t t i t e t a l . was  The  marine  interesting antibiotic activity.  invertebrates.  (NCI)  of  i n v e s t i g a t e d , however, u n t i l r e c e n t y e a r s v e r y  been conducted on the c h e m i s t r y  chemical  the  Products Chemistry  biological  exhaustively had  the s e v e r a l months o f  i n t o a z o o i d w i t h i n hours.  Natural  The  p r i o r to s e t t l i n g .  feed during  displayed  Cancer  Insti-  system) or Walker  Among the organisms t e s t e d  Bugula n e r i t i n a (bryozoa),  c o l l e c t e d o f f the  by  Northeast  c o a s t o f the G u l f o f Mexico. The  anticancer  studies  n e r i t i n a c u l m i n a t e d i n 1982  i n i t i a t e d i n 1968 w i t h the  by P e t t i t  i s o l a t i o n and  o f a remarkable a n t i n e o p l a s t i c compound named s t r u c t u r e o f t h i s compound, which was from a bryozoan, was  s o l v e d by  the  et  al.  B.  structure elucidation  bryostatin  1  f i r s t pure m e t a b o l i t e  a combination o f  on  x-ray  (1).^  The  isolated  crystallographic  4a-  and  spectroscopic  techniques.  B r y o s t a t i n 1 (1)  was a c t i v e a g a i n s t the  murine P388 l y m p h o c y t i c leukemia (PS system) both i n v i v o and i n Subsequent  studies  on  the sea-mat l i k e B^. n e r i t i n a have y i e l d e d  s t r u c t u r a l v a r i e n t s on the same b r y o p y r a n m a c r o l i d e s k e l e t o n in  bryostatin  (5) , 1 0  6 (6)  1 1  1  (1).  They a r e b r y o s t a t i n s 2 ( 2 ) ,  and 7 ( 6 a ) .  7  1 0  R=  H  o  1a  vitro.  R ' = H  4  (la)  3 (3) , 8  four found  4 (4) ,5 9  -4b-  - 5 -  5  R= C O B u , R= A c  6  R= C O P r , R= A c n  A simultaneous (bryozoan)  6a  also  study  R=COCH  3  , R=H  7 R'= R"= C O P r  by  Pettit  et  a l . on  Amanthia  n  convoluta  y i e l d e d an e x t r a c t p o s s e s s i n g a n t i n e o p l a s t i c a c t i v i t y .  A. c o n v o l u t a . which was found  t o grow t o g e t h e r  with  B^. n e r i t i n a  in  a  p a r a s i t i c o r e p i p h y t i c - l i k e manner, has y i e l d e d the same group o f bryostatins It  is  (1-6) as w e l l as a new compound d e s i g n a t e d unclear  convoluta convoluta.  or  whether  brystatin  transferred  from  8  is  a  JL. n e r i t i n a  bryostatin  8 (7).^  genuine c o n s t i t u e n t o f A. and  concentrated  by  A.  - 6 Still  t o be determined  i s whether these compounds o r i g i n a t e from de  novo b i o s y n t h e s i s o r from a common  from  source  such  as  bacteria  or  P e t t i t has p o s t u l a t e d t h a t the i s o l a t i o n o f b r y o s t a t i n 4  phytoplankton. (4)  food  neritina collected  i n such d i v e r s e g e o g r a p h i c a l areas as the  G u l f o f Mexico and the G u l f o f Sagami, Japan i n d i c a t e s t h a t the macrocyclic  l a c t o n e s a r e b i o s y n t h e t i c p r o d u c t s r a t h e r than o f  However,  this  biosynthetic  1 4  can  only  C-acetate  dietary  origin.  be answered by f i n d i n g a d i e t a r y source o r by  feeding s t u d i e s .  C h r i s t o p h e r s e n and C a r l e ,  1 3  i n a series of a r t i c l e s s t a r t i n g i n  1978  r e p o r t e d the i s o l a t i o n o f monoterpenes and n i n e new b r o m o - a l k a l o i d s  from  the bryozoan initiated prior  study  Flustra foliacea.  by  an  Their  work  with  marine  bryozoa  i n t e r e s t i n the study o f c h e m i c a l m e s s e n g e r s .  carried  marine  ' t r a n s m i t t e r s ' , i t was r e p o r t e d t h a t o l d e r f r o n d s o f F\_ f o l i a c e a  emitted  ents  of  the  by  Al-Ogily  and  Knight-Jones ^  In a  on  a l e m o n l i k e odor.  out  14  was  1  T h i s odor was a t t r i b u t e d t o the monoterpene c o n s t i t u -  bryozoan,  citronellol  (10), n e r o l  8  9  namely,  cis-citral  (11) and g e r a n i o l  1 0  (12).  1 1  (8),  trans-citral  1 6  1 2  (9),  - 7 The nine novel bromo-alkaloids isolated either  the  physostigmine,  from  F\_ f o l i a c e a  possess  indole, or quinoline r i n g systems.  A l l the  structures were solved by spectroscopic means. and  Carle  Christophersen  reported the i s o l a t i o n and structure e l u c i d a t i o n of f l u s t r a -  mine A (13) and flustramine B (14), both bromophysostigmine ent.  In 1979,  skeleton  possessing  the  unprecedented  and a rare N-8 y,7-dimethylallyl  substitu-  1 7  13  Christophersen foliacea  14  and Carle, i n a continuation  ( L . ) , reported  the  flustramine C (15), flustraminol  isolation A  and  (16) and  of  the  study  of F.  structure e l u c i d a t i o n of flustraminol  B  (17).^  These three structures possess the basic 6-bromo physostigmine skeleton, however, i n contrast to compounds (13) and (14) they one  isoprene  substituent.  a l l contain  only  In 1981, Wulf et a l . reported the i s o l a t i o n  - 8 -  and  s t r u c t u r e e l u c i d a t i o n o f a bromo-indole  (18).1^  derivative,  A d d i t i o n a l J\ f o l i a c e a metabolites  flustrabromine  include flustramide A  6-bromo-N-methyl-N-formyltryptamine  (20)^  ethyl)quinoline  i s o l a t e d n a t u r a l l y o c c u r r i n g bromo-  quinoline.  A  (21),21 series  dihydroflustramine  of  C  the new  (22),  first  strongly flustramine  and  (19),  u  7-bromo-4-(2-ethoxy-  antimicrobial D  N-oxide (24), f l u s t r a m i n e D N-oxide (25) and  bromoalkaloids,  (23), d i h y d r o f l u s t r a m i n e  i s o f l u s t r a m i n e D (26), were  r e c e n t l y i s o l a t e d from F l u s t r a f o l i a c e a c o l l e c t e d i n the Bay Chartelline  A  (27),  a  novel  was  chartelline A  reported (27) was  crystallography.  i n 1985  o f Fundy.22  p e n t a h a l o g e n a t e d a l k a l o i d from  b r y o z o a n C h a r t e l l a papyracea. which b e l o n g s to the foliacea.  by Chevolot  C  same  e t al.23  unambiguously a s s i g n e d by  family The  single  as  the F.  s t r u c t u r e of  crystal  X-ray  10 -  - 11 -  Two (28) from  gramine d e r i v e d b r o m o - a l k a l o i d s ,  and i t s N-oxide the  (29),  subtropical  2,5,6-tribromo-N-methylgramine  were i s o l a t e d by Sato bryozoan  and  Fenical  Zoobotryon v e r t i c i l a t u m .  N-oxides are commonly found a l o n g w i t h f r e e a l k a l o i d s s o u r c e s , however, t h e i r o c c u r r e n c e i n F\. f o l i a c e a  0  29  2 2  from  2 4  in  1983  Alkaloid terrestrial  and Z^. v e r t i c i l a t u m ' 2  12 -  appear  to be the  Initial  only  bioassays  fertilized  sea  such  show  urchin  reported  that  (ED50  group  =16  contact  dermitis  gelatinosum.^°  Dogger  caused  by  Sulfoxonium  Bank  exposure  ions  had  from  marine  inhibits  ug/mL).  i n 1977.^  (2-Hydroxyethyl)dimethylsulfonium be the c a u s a t i v e agent o f  (28)  compound  eggs  s y n t h e s i z e d by an I t a l i a n  examples  sources.  cell division (28)  Compound  of was  5  i o n (30), has been determined to itch, to not  an the  eczematous bryozoan  previously  allergic  Alcyonidium  been  found  in  nature.  0"  CH  I  3  C H  / 3  C H — C H — OH 2  2  * 30  Nudibranchs  are  extracts of three Tambi e abdere. biologically compounds  of  the main bryozoan p r e d a t o r s .  nudibranchs,  have  Roboastra t i g r i s .  a l l been  active  found  bipyrroles,  to  contain  tambjamines  A-D  Methanolic  Tambje e l i o r a the  same  (31-34).^  and  group  of  These  were a l l t r a c e d to a d i e t a r y s o u r c e , the b r y o z o a n S e s s i b u g u l a  translucens. translucens  one  Tambjamines A-D and  compounds f o r s e l f  it  is  are the major secondary m e t a b o l i t e s o f  believed  defence.  When  the  nudibranchs  FL. t i g r i s .  the  use large  S.  these d i e t a r y carnivorous  13 -  33  nembrothid  34  nudibranch  T. abdere and Tj. e l i o r a yellow  mucus  from  A-D.  the  goblet  b r e a k o f f the a t t a c k . tambjamines  attacks  the  two s m a l l e r nembrothid n u d i b r a n c h s  following  occurs;  The s e c r e t i o n has been shown  Tj_ e l i o r a  motion.  In  produces  a  c e l l s i n the s k i n which causes R^. t i g r i s t o  does  not  appear  s e c r e t i o n , however, i t d i d attempt to escape writhing  T\_ abdere  subsequent  laboratory  to  contain  mainly  t o produce a d e f e n s i v e tigris  by  observations  a  vigorous  i t has been  - 14 shown t h a t |L_ t i g r i s p r e f e r r e d t o e a t Tj. e l i o r a r a t h e r In  than  L  f u r t h e r b i o a s s a y s t u d i e s , these b i p y r r o l e s have d i s p l a y e d  abdere.  antimicro-  b i a l a c t i v i t y against various bacteria. Fusetani et a l . microbial  2 8  r e c e n t l y r e p o r t e d the i s o l a t i o n o f a  tetrapyrrole  35  blue  from the b r y o z o a n Bugula d e n t a t a .  35 was p r e v i o u s l y found i n a mutant  strain  of  the  bacteria  anti-  Compound Serratia  marcescens.  A  new  p u r i n e d e r i v a t i v e , p h i d o l o p i n (36), was r e p o r t e d In 1984 by  Ayer and A n d e r s e n the  waters  2 9  from the b r y o z o a n P h i d o l o p o r a p a c i f i c a c o l l e c t e d  o f f B r i t i s h Columbia.  i t s desmethyl d e r i v a t i v e methyl-2-nitrophenol  C o - o c c u r r i n g w i t h t h i s compound was  ( 3 7 ) , as w e l l as two  (38)  and  nitrophenols,  4-hydroxy-  4-methoxymethyl-2-nitrophenol  P h i d o l o p i n was shown t o p o s s e s s p o t e n t i n - v i t r o a n t i m i c r o b i a l and algal activities.  in  (39). anti-  -  15  -  - 16 B.  SYNTHESIS OF PHIDOLOPIN AND DESMETHYLPHIDOLOPIN  The t o t a l s y n t h e s i s o f p h i d o l o p i n (36) and d e s m e t h y l p h i d o l o p i n (37) was u n d e r t a k e n  i n o r d e r t o c o n f i r m t h e s t r u c t u r e o f 37 p r o p o s e d by  Ayer  99  et  al. *  i n t h e i r 1984  s t u d y o f the c h e m i s t r y o f Pj. p a c i f i c a . and a l s o  t o d e v i s e an e f f i c i e n t method f o r p r o d u c i n g both metabolites f o r f u r t h e r b i o l o g i c a l  The  synthetic  i s shown i n Scheme 1. protection  sufficient  testing.  p l a n f o r t h e t o t a l s y n t h e s i s o f these two compounds The f i r s t s t e p  of  the  synthesis  entailed  the  o f t h e h y d r o x y l f u n c t i o n a l i t y on 4 - m e t h y l - 2 - n i t r o p h e n o l  w i t h a base s t a b l e p r o t e c t i n g group, w h i c h c o u l d be the  quantities of  f i n a l step i n synthesis.  easily  removed  P r o t e c t i n g the phenol as i t s m e t h y l  o r as i t s methoxymethyl e t h e r (MOM)  appeared  to  be  the  (40)  most  as  ether direct  approach. The  f o l l o w i n g methods  were  attempted  p r o t e c t i o n i n the g r e a t e s t y i e l d (Scheme 2).  t o a c c o m p l i s h the d e s i r e d M e t h y l a t i o n o f 4-methyl-2-  -  ieme 1:  Synthetic plan for phidolopin phidolopin  36 R=CH  3  37  17  R=H  (37)  (36) and  desmethyl-  18 -  Scheme 2:  Protection  of 4-methyl-2-nitrophenol  40  a  b  c  40  nitrophenol potassium  (40) was carbonate  (Scheme 2 a ) . ^ (1:10  u  a c h i e v e d by w i t h 1.5  reacting  40  in  e q u i v a l e n t s o f methyl  acetone  and  excess  i o d i d e under r e f l u x  Continuous m o n i t o r i n g by t h i n l a y e r chromatography ( T L C )  e t h y l a c e t a t e / p e t r o l e u m e t h e r ) showed r a p i d f o r m a t i o n o f a new  a b s o r b i n g component a t Rf 0.13 material  a t Rf 0.35.  which was  more p o l a r  than  the  A f t e r 2.5 h r , i t appeared by t i c t h a t the  UV  starting reaction  - 19  had proceeded tioning  to completion.  between  distilled  organic layer with Na2S0^, heavy  brown  Work-up o f the r e a c t i o n mixture by  oil.  water  and  filtering  Purification  dichloromethane,  NMR  (Figure  showed the r e q u i r e d (s,  3H),  of  t h i s o i l by p r e p a r a t i v e TLC  methods,  but u s i n g two preparation the f i r s t diethyl  solution  of  t o r e d by TLC  was  mixture  using  of  were  The  f o r 48 h r . The  A new  work-up  of  (yield  a t 6 3.95  ppm  (Scheme 3 ) .  as  approaches  (Scheme 2b,  c).  to 3  1  prepared  dropwise  at  r e a c t i o n , which was was  more p o l a r UV involved  0°C  to  ,  3  the 2  in  ppm  o f 42  allowed  the  red  proceed  at  a b s o r b i n g component  (Rf  to  partitioning  the  reaction  unreacted  over  preparative  y i e l d e d 42 as a c l e a r y e l l o w o i l ( y i e l d  ( F i g u r e 2) showed resonances  ( s , 2H)  dry  c o n t i n u a l l y moni-  and e v a p o r a t i o n i n vacuo. P u r i f i c a t i o n by  ^-H NMR  In  Chloro-  water and d i e t h y l e t h e r , d r y i n g the o r g a n i c l a y e r  a s s i g n e d t o the MOM  42 showed a p a r e n t i o n a t m/z  reaction.  derivative  added  The  e t h y l acetate/hexanes)  and 6 5.25  amount  167  explored  e t h y l acetate/hexanes),  formed.  (1:10  oil  a s u s p e n s i o n o f sodium h y d r i d e ( e x c e s s ) .  phenoxide.  (1:10  Na2S04, f i l t e r i n g  3H),  bases,  (1.5 equiv) was  the  between  36.3%).  f o r the OCH3 p r o t o n s  p a r e n t i o n a t m/z  -OCH2CH3 (MOM)  the  room temperature  TLC  resonance  case, the phenoxide o f n i t r o p h e n o l (40) was ether  (2:10  b o t h i n v o l v i n g the use o f c h l o r o m e t h y l methyl e t h e r ,  methylmethylether  0.17)  NMR  different of  yellow  a  1) and mass s p e c t r o m e t r i c a n a l y s i s o f t h i s o i l  ( T a b l e 1) and a MS  Two  d r y i n g o f the  and e v a p o r a t i o n i n vacuo y i e l d e d  e t h y l a c e t a t e / p e t r o l e u m e t h e r ) gave 41 as a v i s c o u s 71%).  parti-  197  a t 6 3.53  group ( T a b l e 1 ) .  as r e q u i r e d (Scheme  s t a r t i n g m a t e r i a l 40 was  4).  ppm  (s,  A  EIMS  A  a l s o r e c o v e r e d from  large this  - 22 -  Table  1:  80 MHz  i  H  NMR  data on p r o t e c t e d n i t r o p h e n o l s 41 and  Chemical s h i f t , H on C  (CDCI3)  41  #  42  3  7.65  (d, J - 2 Hz,  5  7.32  (dd, J = 9,  6  6.97  (d, J •=  7  2.35  8  3.95  9  6 ppm  42  IH)  7.56  (d, J - 2 Hz,  7.30  (dd, J = 9,  7.13  (d, J •= 9 Hz,  ( s , 3H)  2.35  (s,  3H)  ( s , 3H)  5.25  (s,  2H)  3.53  (s,  3H)  2 Hz,  8 Hz, IH)  IH)  IH)  2 Hz,  IH)  IH)  23 Scheme 3:  I n t e r p r e t a t i o n o f the MS f r a g m e n t a t i o n o f 41  m/z 105 (27°/ ) 0  0CH  "1* /^N0 /-CH #  3  2  NO  2  -NO  m/z  3  137 (49 •/.)  U0  2  41  m/z 120 ( 82 %)  m/z 167(78%)  Scheme 4:  I n t e r p r e t a t i o n o f t h e MS f r a g m e n t a t i o n o f 42  I"I  —  0CH,0CH  3  —  m  »  /  z  ' &<t' X ) 3  m/z 167 ( 37 %)  OCH: 42 m  /  z  m/z 197( 55%)  166(3%)  - 24 -  In an attempt t o d e v i s e a h i g h e r y i e l d i n g and the  preparation  of  the  MOM  derivative  carbonate and 6 e q u i v a l e n t s  reacted with these  the n i t r o p h e n o l  conditions,  o f the c h l o r o m e t h y l tate/hexanes).  nent  carbonate.  of chloromethyl  40 i n acetone a t room  for  the r e a c t i o n Thus,  excess  methyl e t h e r were  temperature.  Under  the r e a c t i o n went almost s p o n t a n e o u s l y upon a d d i t i o n methyl e t h e r as i n d i c a t e d by TLC  There  (1:10  ethyl  ace-  appeared t o be much l e s s s t a r t i n g m a t e r i a l a f t e r  10 t o 15 minutes than t h e r e was i n the Following  method  42, we m o d i f i e d  c o n d i t i o n s t o use the m i l d e r base, potassium potassium  cleaner  previous  method  work-up and p u r i f i c a t i o n conducted as b e f o r e ,  after  48  hr.  a s i n g l e compo-  ( y i e l d 73%) was i s o l a t e d , which had i d e n t i c a l s p e c t r a l f e a t u r e s  to  the p r e v i o u s l y p r e p a r e d MOM p r o t e c t e d n i t r o p h e n o l 42. Lister production  et  al.  3 3  have l o o k e d  a t the o p t i m i z a t i o n o f c o n d i t i o n s f o r  o f 7 - b e n z y l x a n t h i n e (43) and  9-benzylxanthine  (44)  deriva-  t i v e s v i a the N7 and N9 a l k y l a t i o n o f x a n t h i n e s .  0  44  43  They  found t h a t 7 - ( 4 - n i t r o b e n z y l ) t h e o p h y l l i n e  benzyl)theophylline  (50) c o u l d be produced by  (48) o r 7-(4-methyl-  refluxing  4-nitrobenzyl-  - 25 -  bromide  (47)  or  4-methylbenzylbromide  aqueous sodium h y d r o x i d e Lister's  work  synthesis of conversion  as  a  model,  phidolopin of  the  (.1 M)  (36)  protected  (49) w i t h t h e o p h y l l i n e (45) i n  f o r 2 hours  carried  out  (Scheme 7 ) . The equiv)  with  of  and  desmethylphidolopin  N-bromosuccinamide  (37)  was  the  agents.  (NBS)  (41) as  was  successfully  the b r o m i n a t i n g  a  c a r r i e d out by d i s s o l v i n g compound 41 and  agent  NBS  minimum amount o f d i s t i l l e d carbon t e t r a c h l o r i d e and of l i g h t  A l k y l a t i o n o f t h e o p h y l l i n e (45) w i t h  (150 W t u n g s t e n  47  Base Br  45  Using  3 4  r e f l u x i n g t h i s s o l u t i o n i n the presence  Scheme 5:  6).  n i t r o p h e n o l s 41 and 42 t o b e n z y l bromide  4-methoxy-3-nitrotoluene  r e a c t i o n was in  and  i t seemed t h a t a l o g i c a l next s t e p i n the  d e r i v a t i v e s which c o u l d a c t as a l k y l a t i n g Bromination  (Schemes 5  47  48  (2 then  bulb).  26 Scheme 6:  A l k y l a t i o n of theophylline with  49  - 27 The  reaction  was  monitored  very  c l o s e l y by TLC (3:10 e t h y l  hexanes) t o ensure t h a t o n l y monobromination minutes,  a  new  more  polar  occurred.  indicating  the f o r m a t i o n o f bromine.  to  a  up.  be the  reddish  5  As time brown,  and  carefully  R a p i d p u r i f i c a t i o n o f the new component by p r e p a r a t i v e TLC  (3:10 e t h y l a c e t a t e / h e x a n e s ) i n a darkened room, a f f o r d e d ( y i e l d 45%),  only  When a s u f f i c i e n t amount  o f the new component had formed, the r e a c t i o n was h a l t e d worked  After  component became apparent on TLC.  proceeded, t h e r e a c t i o n mixture went from y e l l o w perhaps  acetate/  a  yellow o i l  which was i d e n t i f i e d by low r e s o l u t i o n mass s p e c t r o m e t r y to  the d e s i r e d b e n z y l bromide 51 (Scheme 8 ) .  NMR was n o t u s e f u l  in  c h a r a c t e r i z a t i o n o f 51 due t o t h e r a p i d d e c o m p o s i t i o n o f t h i s h i g h l y  labile  substance.  Scheme 8:  MS f r a g m e n t a t i o n o f compound 51  (25 V.)  *  51  m/z  2 4 5 / 247(2,3 V . )  m/z 166 (657.)  - 28 -  The in  bromination  o f the MOM  p r o t e c t e d n i t r o p h e n o l 42 was c a r r i e d out  a s l i g h t l y m o d i f i e d manner (Scheme 9 ) .  Compound 42 was d i s s o l v e d i n  a minimum amount o f carbon t e t r a c h l o r i d e and the s o l u t i o n was brought to r e f l u x while being  i r r a d i a t e d w i t h a 220 W sun lamp.  Two e q u i v a l e n t s o f  NBS i n carbon t e t r a c h l o r i d e were s l o w l y added t o the r e f l u x i n g s o l u t i o n . The  r e a c t i o n was c a r e f u l l y monitored by TLC (3:10 e t h y l acetate/hexanes)  to ensure t h a t o n l y monobromination  Scheme 9 :  Bromination  occurred.  o f MOM p r o t e c t e d n i t r o p h e n o l 42  0M0M  0M0M  When the r e a c t i o n was l e f t tion  of  t o r u n f o r more than 20 minutes,  The  monobrominated  product  52  v i s i b l e on TLC w i t h i n 5 minutes and the r e a c t i o n was complete by 15  minutes. avoid  forma-  p o l y b r o m i n a t e d s p e c i e s would occur as i n d i c a t e d by the appear-  ance o f numerous more p o l a r TLC s p o t s . was  the  The r e a c t i o n was t e r m i n a t e d  by removing the l i g h t source.  the l o s s o f m a t e r i a l due t o decomposition, the brominated  was never  isolated  for characterization,  d i r e c t l y i n the a l k y l a t i o n r e a c t i o n s .  but  instead  To  species  i t was  used  Even though 52 was n o t c h a r a c t e r -  - 29 -  i z e d s p e c t r o s c o p i c a l l y , TLC showed 52 t o be s l i g h t l y more p o l a r than  the  starting  material  42 ( R  f  0.34)  (3:10 e t h y l  (Rf 0.28)  acetate/hexanes)  s i m i l a r t o the p o l a r i t y d i f f e r e n c e s between 41 and 51. A l k y l a t i o n was c a r r i e d out w i t h both the methyl as w e l l as the protected  a l k y l a t i n g agents.  R e a c t i o n o f 51 w i t h t h e o p h y l l i n e  o f r e f l u x i n g 0.1 M sodium h y d r o x i d e and 3 mL t e t r a h y d r o f u r a n gave  both  the  desired  N7  alkylated  a l k y l a t e d compound 54 (Scheme 1 0 ) . could  be  The N7  and  d i s t i n g u i s h e d by examination o f the  compounds (Table  Scheme 10:  compound  2).  53 N9 NMR  MOM  i n 1.5 mL for  2  hr  as w e l l as the N9 alkylated  products  s p e c t r a o f the two  The c h e m i c a l s h i f t o f the b e n z y l i c p r o t o n s i n the  A l k y l a t i o n of theophylline  (45) w i t h 51  - 30 -  T a b l e 2:  80 MHz  i  H  NMR  data comparison o f 53 and  Chemical H on C o r  1'  N  shift,  6  54  ppm  53  #  54  5..48  (s,  2H)  5..53  (s,  2H)  NI  Me  3..43  (s,  3H)  3..39  (s,  3H)  N3  Me  3..58  (s,  3H)  3..56  (s,  3H)  3'  7,.38  (dd, J •= 9,  7,.58  (dd, J - 9,  4'  7 .00  (d, J -= 9  Hz, IH)  7,.09  (d, J = 9  Hz, IH)  7'  7..68  (d, J •  2  Hz, IH)  7 .89  (d, J •= 2  Hz, IH)  2 Hz,  IH)  4'  NO  2 Hz,  IH)  - 31 -  N7 a l k y l a t e d s p e c i e s was found t o be 6 5 . 4 8 ppm  2H),  whereas  the N9 a l k y l a t e d compound r e s o n a t e d a t 6* 5 . 5 3  benzylic  protons  ( s , 2H).  A l s o a l t e r e d were the s h i f t s o f the aromatic p r o t o n s  in  2.  Table  in  (s,  Comparison o f the c h e m i c a l s h i f t s o f the b e n z y l i c  as  the ppm seen  protons  to the observed v a l u e s f o r p h i d o l o p i n (36) a l l o w e d the assignment o f the correct In  structures. the  case o f the MOM  d e r i v a t i v e 42, the b r o m i n a t i o n and a l k y l a -  t i o n r e a c t i o n s were c a r r i e d out w i t h o u t (Scheme  11).  Isolation  of  intermediate  F o l l o w i n g the b r o m i n a t i o n r e a c t i o n , the r e a c t i o n m i x t u r e  was b r i e f l y c o o l e d and the succinamide was f i l t e r e d o f f .  Scheme 11:  52  B r o m i n a t i o n and a l k y l a t i o n  5 5 R=CH 56  R=H  32  The  resulting  dryness b e f o r e of  being  theophylline  stirring used  filtrate  was  c a r e f u l l y e v a p o r a t e d i n vacuo to near  d i s s o l v e d i n THF  (45)  i n 0.1  -  and  added dropwise to  The  i n the a l k y l a t i o n r e a c t i o n assumed a 100% As  reaction  a r e s u l t , excess t h e o p h y l l i n e  mixture.  solution  M sodium h y d r o x i d e s o l u t i o n which had  f o r 20 minutes a t room temperature.  reaction.  a  amount o f  yield  (45)  been  theophylline  i n the  bromination  always appeared i n  the  A f t e r 18 hours o f s t i r r i n g a t room temperature,  TLC  a n a l y s i s showed the p r e s e n c e o f the p r o t e c t e d n i t r o p h e n o l 42 as w e l l  as  a new  more p o l a r component.  (3:10  e t h y l a c e t a t e / h e x a n e s ) y i e l d e d the p o l a r c o n s t i t u e n t  solid  (yield  25%)  Work-up and p u r i f i c a t i o n by p r e p a r a t i v e  which  was  spectrometric  analyses  to be  features  the  NMR  of  5.46  ppm  assigned  ppm  assigned  i o n a t m/z reaction  by  NMR  group.  The  p r o v e d to be  a  white  ( F i g u r e 3) and mass  spectrum were a sharp two  54.  resonances a t S 3.51  The  yield  in  Key  s i n g l e t at S  proton  and  mass spectrum o f 55 showed a  (8% o f base) (Scheme 12).  (32%)  ^H  the d e s i r e d p r o t e c t e d p h i d o l o p i n  to b e n z y l i c p r o t o n s and  to the MOM  375  n e a r l y 50%  ^H  shown  as  TLC  this  5.25 parent  two  step  the lowest i n the t o t a l s y n t h e s i s , however,  o f the p r o t e c t e d n i t r o p h e n o l 42 was  recoverable  by  chromato-  a l k y l a t i o n , MOM  protected  g r a p h i c means a f t e r work-up o f the r e a c t i o n . Using  the same method o f b r o m i n a t i o n and  desmethylphidolopin (24.7%)  by  (Scheme 11). dence N9  o f NI  (56)  was  substituting As b e f o r e , or N9  synthesized  3-methylxanthine  o n l y the N7  a l k y l a t i o n o f x a n t h i n e s c o u l d be  nearly  (46)  the  same  yield  for theophylline  p r o d u c t was  a l k y l a t i o n ( F i g u r e 4) .  t i o n i n a more c o n c e n t r a t e d  in  formed,  with  no  (45) evi-  L i s t e r - * found t h a t e x c l u s i v e  achieved  3  by c a r r y i n g out  the  alkyla-  b a s i c s o l u t i o n under r e f l u x c o n d i t i o n s f o r 3  F i g u r e 4:  80 MHz  i  H  NMR  o f MOM  protected desmethylphidolopin  56  35  Scheme 12:  MS  hours w h i l e C-8 for  4  hours  -  f r a g m e n t a t i o n o f 55  a l k y l a t i o n would occur i n dimethylformamide  (Scheme 13).  I t appears  t h a t the N7  the k i n e t i c a l l y p r e f e r r e d p r o d u c t w h i l e the N9 dynamically previously  preferred in  nitrophenol 9).  the  alkylation  alkylation  of  products  theophylline  the use o f m i l d e r  motes the a l k y l a t i o n o f o n l y one p o s i t i o n , Deprotection  for  the  are  the  of  with  the  reaction  (Scheme  conditions  pro-  N7.  the m e t h y l a t e d p h i d o l o p i n d e r i v a t i v e 53 proved to 3  of  seen  methylated  U s i n g the method o u t l i n e d by Olah e t a l . , ^ which  cleavage  thermo-  t h e o p h y l l i n e . As  41, r e f l u x c o n d i t i o n s y i e l d e d a mixture o f p r o d u c t s  T h e r e f o r e , i t appears  be d i f f i c u l t .  a l k y l a t i o n product i s  and C8 of  under r e f l u x  methyl  allows  e t h e r s by treatment w i t h c h l o r o t r i m e t h y l -  - 36 -  Scheme 13:  A l k y l a t i o n of theophylline  (45) under v a r i e d  conditions  s i l a n e and sodium i o d i d e i n d r y a c e t o n i t r i l e a t room temperature f o r 8 to 10 hours, y i e l d e d o n l y s t a r t i n g m a t e r i a l Deprotection  of  the MOM  gave h i g h y i e l d s o f p r o d u c t . for acid  one 3 7  hour  plus  in  (Scheme 14).  d e r i v a t i v e s proved t o be much s i m p l e r and Refluxing  e i t h e r MOM  a minimum amount o f c h l o r o f o r m  one drop o f c o n c e n t r a t e d  sulphuric acid  d e r i v a t i v e 55 containing yielded  or  56  50% a c e t i c phidolopin  - 37 -  Scheme 14:  D e p r o t e c t i o n o f 53 w i t h c h l o r o t r i m e t h y l s i l a n e  (36)  desmethylphidolopin  and  (37)  respectively  p u r i f i c a t i o n by p r e p a r a t i v e TLC ( F i g u r e s 5, 6 )  Scheme 15:  R=CH  56  R=H  3  3)  (Scheme 15).  D e p r o t e c t i o n o f 55 and 56 w i t h d i l u t e  55  (Table  acid  36  R=CH  37  R=H  3  after  F i g u r e 5:  80 MHz  1  H  NMR  of phidolopin  36  - 40 -  T a b l e 3:  80 MHz  ^-H NMR  phidolopin  data f o r p h i d o l o p i n (36) and  (37)  Chemical H on C or N  desmethyl-  shift,  5  ppm  36  #  37  11.03  NI--H  (s,  IH)  -  NI Me  3.,59  (s, 3H)  N3 Me  3. 59  (s, 3H)  3.46  (s,  3H)  8  7..65  ( s , IH)  7.82  (s,  IH)  1'  5..48  (s, 2H)  5.45  (s,  2H)  3'  7..63  (dd, J - l . 9 , 8 . 5 Hz,  7.68  (dd, J-8.5,1.9 Hz,  4'  7,.16  (d, J - 8.5  Hz,  IH)  7.10  (d, J - 8.5  Hz,  IH)  7'  8 .06  (d, J = 1.9  Hz,'  IH)  8.05  (d, J = 1.9  Hz,  IH)  36  IH)  37  IH)  - 41 Spectroscopic  data  p r o d u c t s was i d e n t i c a l natural  products.  (Table to  the  3)  obtained  for  spectroscopic  data  the  two  synthetic  recorded  f o r the  The t o t a l s y n t h e s i s o f p h i d o l o p i n (36) was r e p o r t e d oo  by a Japanese g r o u p syntheses  of  author  1985.  in  assignment  of  spectrum  (Table  4).  s i m u l t a n e o u s l y w i t h the c o m p l e t i o n  phidolopin In the  t h i s d a t a and the  J O  NMR of  1 3  (36)  addition C  NMR  and to  desmethylphidolopin the  spectrum  synthesis was  (37)  the  (Figure  7),  total  ( 3 7 ) ^ by t h i s 3  compound  reported (Table 4).  d a t a o f compound (36) ( F i g u r e  desmethylphidolopin  of  of  (36), Using  assignment  8) was  of  simplified  - 44 -  T a b l e 4:  1 J  C  NMR  data comparison f o r 3 6  j e  and 37  Chemical s h i f t ,  C  36  #  a  5 ppm  37  b  -  NI Me  28. 0 (q)  N3 Me  29. 7 (q)  28. 5 (q)  2  155.,3 (s)  154.,9 (s)  4  149..3 (s)  150..4 (s)  5  106..8 (s)  106..3 <s)  6  151..6 (s)  151,.1 (s)  8  140..6 (d)  142,.3 (d)  1'  49..0 ( t )  47,.9 ( t )  2»  128 .1 (s)  127,.6  3'  124 .5 or 121.0 (d)  119 .9 o r 125.2 (d)  4'  133..7 (s)  135,.2 (s)  5'  155,.2 (s)  152,.8 (s)  6'  124..5 o r 121.0 (d)  7'  137..0 (d)  a  100  b  75  MHz, MHz,  CDC1 CDCI3  3  +  DMS0-d  6  O)  . 119,.9 or 125.2 (d) 136..3 (d)  - 45 -  C.  The pacifica  NITROPHENOLS FROM NORTHEAST PACIFIC BRYOZOANS  collected  Ayer e t a l . ^ 9 i of  (36),  i s o l a t i o n of phidolopin  fouling  from  the b r y o z o a n  Phidolopora  o f f the c o a s t o f B r i t i s h Columbia, was r e p o r t e d by  1984.  A t t e n t i o n was drawn t o t h i s bryozoan by t h e l a c k  organisms  on i t s s k e l e t o n as w e l l as by t h e s t r o n g i n v i t r o  n  a n t i - a l g a l and a n t i - b a c t e r i a l a c t i v i t y d i s p l a y e d by i t s crude F u r t h e r i n v e s t i g a t i o n o f P_j_ p a c i f i c a due  ( r e f e r r e d t o as the " l a c y bryozoan"  t o i t s i n t r i c a t e c a l c i u m carbonate  ruffled  (37),  which  4-hydroxymethyl-2-nitrophenol  4-methoxymethyl-2-nitrophenol  resembles  (39),  discovery  metabolites  o t h e r common  the  later  believed  (38) to  of  interesting  i n Pj. p a c i f i c a .  Northeastern  Pacific  biologically  metabolites.  bryozoans  Crude  A l l new bryozoan specimens methanol  vitro antibacterial,  be  an  which  were c o l l e c t e d  a n t i f u n g a l and a n t i a l g a l  live  in  by  hand  similar active using  activity.  (d'Orbigny 1852), u s u a l l y  the "Southern Staghorn" bryozoan,  Classified  referred  t o as  i s commonly found on o f f s h o r e r e e f s as  w e l l as i n t e r t i d a l r o c k s (-2 t o -10 m) from the B r i t i s h Baja, C a l i f o r n i a .  active  e x t r a c t s o f these animals were assayed f o r i n -  Diaperoecia c a l i f o r n i c a  to  and  we i n i t i a t e d an examination o f  h a b i t a t s w i t h the hope o f d i s c o v e r i n g a d d i t i o n a l new b i o l o g i c a l l y  SCUBA.  a  artifact.  Encouraged by the secondary  exoskeleton  l a c y network) l e d t o the i s o l a t i o n o f o t h e r n o v e l n i t r o p h e n o l s ;  desmethylphidolopin  isolation  extracts.  Columbia  coast  i n the bryozoan o r d e r C y c l o s t o m a t a , D.  c a l i f o r n i c a p o s s e s s e s an i n f l e x i b l e c a l c i u m carbonate e x o s k e l e t o n  which  - 46 -  is  coral-like  in  appearance,  with  each c o l o n y a t t a i n i n g a h e i g h t o f  a p p r o x i m a t e l y 10 cm and a diameter o f 13 t o 15 cm. fied  by  its  light  yellow  tubular  It is  also  identi-  branches which have a f l a t  cross-  section. D. c a l i f o r n i c a was B.C.  The  bryozoans  first  work-up.  At  in  Barkley  Sound,  (653 g d r i e d weight a f t e r e x t r a c t i o n ) were immedi-  a t e l y soaked i n methanol before  c o l l e c t e d i n June 1984  and r e f r i g e r a t e d  the  end  of  this  decanted o f f and the bryozoans were ground  (-2°C) f o r up t ime, in  to  seven  the methanol a  waring  layer  blender  days was with  -  f r e s h methanol,  and  filtered.  e x t r a c t s were c o n c e n t r a t e d volume and ethyl  The  the r e s u l t i n g aqueous m e t h a n o l i c  acetate.  ethyl  The  acetate  dark r e d o i l . by  combined  The  with  s o l u b l e e x t r a c t was  dried  the  and  g (.89%) o f a  most  each o f which on b i o a s s a y  antibacterial activity.  (36),  which  l o n g wave ( b l a c k ) UV  (1.0 mg  retained  strongly light.  of  graphy on  Sephadex  phidolopin  (37)  A n a l y s i s by  Purifica-  yellow  the LH-20  (3.7 mg,  two  oil  (Table  6)  *H NMR  value  short  wave  (Table 5)  as  phidolo-  211-212).  l e s s p o l a r f r a c t i o n s by column chromato-  (7:3  methanol/chloroform)  .001%) as a y e l l o w  (Scheme 18).  The  1  solid  gave  ( T a b l e 5)  (Scheme .007%)  17),  (4.5  H  and mass s p e c t r a o f  NMR  mg,  desmethyl-  (38)  compounds were i d e n t i c a l to the s p e c t r a o f a u t h e n t i c This general  both  band.  an Rf  c o n f i r m e d the p r e s e n c e o f  as w e l l as 4 - h y d r o x y m e t h y l - 2 - n i t r o p h e n o l yellow  absorbed  .001%), as y e l l o w n e e d l e s (mpt.  Purification  analy-  p o l a r f r a c t i o n by column chromatography on Sephadex  w e l l as mass s p e c t r o m e t r y (Scheme 16) (36)  and  F l a s h chromatography gave t h r e e f r a c t i o n s d i s t i n g u i s h a b l e  s i m i l a r to p h i d o l o p i n  pin  filtered off  a n a l y s i s o f t h i s band i n d i c a t e d a s i n g l e component w i t h  (dark)  original  extracted  LH-20 (7:3 methanol/chloroform) gave a s t r o n g l y TLC  was  e v a p o r a t e d i n vacuo to y i e l d 5.8  s i s a f f o r d e d moderate a n t i f u n g a l and of  methanol  t h i r d the  sodium s u l f a t e was  s i m i l a r chromatographic p o l a r i t i e s ,  tion  layer  red-brown e t h y l a c e t a t e  was  reddish-brown  i n vacuo down to about one  over anhydrous sodium s u l f a t e . the  47  as  a the  materials.  i s o l a t i o n and p u r i f i c a t i o n procedure was  employed  in  the e x a m i n a t i o n o f t h r e e o t h e r bryozoans, namely, H e t e r o p o r a a l a s k e n s i s . Hippodinlosla insculpta. t a b u l a t e d i n Table  13  and  T r i c c e l a r i a ternata  (Section 6).  1  H  NMR  data  with  the  ( F i g u r e s 5, 6,  results 9,  10)  as  - 48 -  well  as  observed  MS  f r a g m e n t a t i o n p a t t e r n s f o r the i s o l a t e d m e t a b o l i t e s can be  i n T a b l e s 5 and 6 and Schemes 16 and 19 r e s p e c t i v e l y .  T a b l e 5:  NMR  d a t a f o r i s o l a t e d n i t r o p h e n o l s 36-37  Chemical s h i f t , H on C o r N  36  #  1  S ppm 37*  s  -  -  NI Me  3..40 (s,  3H)  N3 Me  3 .59  3H)  8  7,.63 ( s , IH)  7..63 ( s , IH)  1'  5 .46 (s,  5..39  (s,  3'  7..61 (dd, J-8.5,2.5 Hz, IH)  7,.59  (dd, J - 9,2 Hz, IH)  4'  7 .17 (d, J - 8.5 Hz, IH)  7,.13 (d, J - 9 Hz, IH)  7'  8,.09  8..06 (d, J - 2 Hz, IH)  80 MHz  (s,  (d, J - 2.5 Hz, IH)  (CDCl -d DMSO) b  3  2H)  3,.51 ( s ,  3H)  2H)  49 Scheme 16:  MS f r a g m e n t a t i o n o f p h i d o l o p i n (36)  m/z 152 (38 V.)  m/z  180 (100 V.)  3 6  m/z Scheme 17:  331 ( U V.)  MS f r a g m e n t a t i o n o f d e s m e t h y l p h i d o l o p i n (37)  •  m/z 166(100 V.)  m/z 123  (45 V.)  HNCO  - 50 -  6:  Table  1  H  NMR  data f o r i s o l a t e d n i t r o p h e n o l s 38-39  Chemical s h i f t , H on  C  6  ppm  38  #  39  3  8.09  (d, J = 2  5  7.58  (dd, J -= 9,  6  7.18  (d, J - 9  7  4.69  (s,  2H)  -  8  ^-OH  10.54  38  (brs,  IH)  Hz, IH)  2 Hz,  Hz, IH)  IH)  8 .10  (d, J = 2  7,.60  (dd, J - 8,  7,.18  (d, J - 8  4..43  ( s , 2H)  3..44  ( s , 3H)  10,.58  (s, IH)  39  Hz, IH)  2 Hz,  Hz, IH)  IH)  - 53 -  Scheme 18:  MS f r a g m e n t a t i o n  o f n i t r o p h e n o l 38  N0  OH  m / z 123 ( 25 V.) 2  r^N^NOo •  m/z  152 ( 11 V.)  'OH 38 m/z  Scheme 19:  169 (100 V.)  MS f r a g m e n t a t i o n  o f n i t r o p h e n o l 39  OH  V ' ^0CH 39  m/z  -0CH  3  183 (59V.)  3  t  m/z 152 (100 V.)  CNOm/z  136 (21V.)  - 54 -  D.  The as  DISCUSSION  i s o l a t i o n of phidolopin (38)  nitrophenols  and ( 3 9 )  (36), desmethylphidolopin (37)  as  i n some combination from the e x t r a c t s o f  f i v e N o r t h e a s t P a c i f i c bryozoans, which a l l b e l o n g t o d i f f e r e n t raises  a  question  about  fore,  normally  have  3  functionalities  such  as  v e r y r e s t r i c t e d taxonomic d i s t r i b u t i o n s .  i t seems u n l i k e l y  bryozoans  genera,  the o r i g i n o f these secondary m e t a b o l i t e s . ^  N a t u r a l p r o d u c t s which c o n t a i n u n u s u a l groups  well  that  each  member  of  the  diverse  nitro There-  group  of  i n v e s t i g a t e d i n t h i s study would e l a b o r a t e t h i s same group o f  n o v e l secondary m e t a b o l i t e s through de novo b i o s y n t h e s i s . A more p r o b a b l e assumption, the  t h a t these n i t r o p h e n o l s are o b t a i n e d by  bryozoans from some s o r t o f d i e t a r y or s y m b i o t i c m i c r o o r g a n i s m such  as a p h y t o p l a n k t e r , b a c t e r i u m o r fungus, i s q u i t e organisms c h a r a c t e r i s t i c f i l t e r  feeding  consistent  with  capabilities.  A s u r v e y o f o t h e r examples o f p u r i n e d e r i v a t i v e s and n i t r o ing  compounds from marine organisms  the  s e r v e s as a  useful  contain-  background  for  s p e c u l a t i o n about the o r i g i n o f the b r y o z o a n m e t a b o l i t e s . Purine  derivatives  based on the x a n t h i n e n u c l e u s a r e g e n e r a l l y o f  Phidolopin (36)  plant origin. xanthine  alkaloid  caffeine  (58),  isolated  isolated  r e p r e s e n t s o n l y the second example from  from  the  a  marine Chinese  source,  the  gorgonian  of  a  f i r s t being Echinogorpia  pseudossapo.^ Marine  organisms  have  also  elaborated  d e r i v a t i v e s not based on the x a n t h i n e n u c l e u s .  a number o f o t h e r p u r i n e Among  the  most  common  - 55 -  are al. the  the 4 1  purine  ribosides  such  as d o r i d o s i n e  r e p o r t e d the i s o l a t i o n o f d o r i d o s i n e Australian  marine  sponge  a  n  <  j  ^  was  HO  OH 59  found  I n 1980, Cook e t -  (1-methylisoguanosine)  Tedania d i g i t a t a .  p r e v i o u s l y r e p o r t e d from the d i g e s t i v e g l a n d Anisodoris n o b i l i s  (59).  of to  This the  compound  dorid  possess  from was-  nudibranch  potent  muscle  - 56 r e l a x a n t , a n t i - i n f l a n u n a t o r y and a n t i - a l l e r g i c a c t i v i t i e s . of  mice  with  a  The  dose e q u i v a l e n t to lg/Kg o f crude sponge e x t r a c t gave  pronounced muscle r e l a x a t i o n and hypothermia which s e r v e d as a and  r e p r o d u c i b l e assay Other  sine  for purifying  biologically  4 3  and  (61)  was  only  (60)  isoguanosine  Diaulula sandiegensis, found  decisive  59.  a c t i v e marine p u r i n e r i b o s i d e s i n c l u d e spongo-  (2-methoxyadenosine)  digitata  injection  4 3  isolated  from  in  sponge  (61), e x t r a c t e d from the d o r i d  I t i s i n t e r e s t i n g to  4 4  the  sandiegensis  C a l i f o r n i a w h i l e specimens from La J o l l a ,  note  that  collected  California  Tedanianudibranch  isoguanosine  from  Monterey,  contained  only  a  as w e l l as 2'-deoxyadenosine (63) were r e p o r t e d  as  s e r i e s of u n r e l a t e d c h l o r i n a t e d acetylenes. -* 4  Adenosine  (62)  the c a r d i o a c t i v e c o n s t i t u e n t s Finally  9-/J-D-arabinosyladenine  of  the (64)  sponge and  Dasychalina  its  cyathina  3'-0-acetyl d e r i v a t i v e  - 57 (65),  previously  i s o l a t e d as cavolini.  known as p o t e n t s y n t h e t i c a n t i v i r a l a g e n t s , have been  natural  products  Compound  4 7  64  was  from  the  the  Italian  first  gorgonian  antiviral  Eunicella  drug used i n the  t r e a t m e n t o f t h e u s u a l l y f a t a l herpes e n c e p h a l i t i s .  P u r i n e d e r i v a t i v e s o t h e r t h a n r i b o s i d e s have a l s o been marine  found  Two 9-methyladenine d e r i v a t i v e s o f d i t e r p e n e s ,  sources.  A ( 6 6 ) and a g e l i n e B ( 6 7 ) , p o s s e s s i n g moderate i c h t h y o t o x i c as antimicrobial sp_. ^ 4  activity  were  F i v e new 9-methyladenine  isolated  from  the  these m e t a b o l i t e s ,  Okinawan  isolated  from  derivatives sea  the P a c i f i c of  from  ageline well  as  sponge A g e l a s  diterpenes  have  been  sponge, A g e l a s n a k a m u r a i F o u r o f  agelasine-A ( 6 8 ) , agelasine-B ( 6 9 ) , agelasine-C  (70)  -  and a g e l a s i n e - D fifth  new  compound  (71)  ageline  +  A  (66).^0 ATPase  These in vitro  antispasmodic p r o p e r t i e s .  70  -  bicyclic  compound a g e l a s i n e - E ( 7 2 ) ,  Na /K -transporting +  a r e based on  58  diterpene  skeletons.  The  was r e p o r t e d a l o n g w i t h the known 9-methyladeninium and a l s o p o s s e s s  salts  inhibit  a n t i m i c r o b i a l and  59  -  72  Hokupurine melanobranchia it  feeds.  (73) as  has been i s o l a t e d w e l l as the c o r a l  I n c o n t r a s t to the o t h e r  marine s o u r c e s , hokupurine  (73)  from  the  nudibranch  Tubastea c o c c i n e a  purine  derivatives  5 1  73  upon which  reported  has no s i g n i f i c a n t b i o l o g i c a l  NH  Phestilla  from  activity.  - 60 This  survey  of  p u r i n e m e t a b o l i t e s from marine sources  n e a r l y a l l the compounds biological  activity.  Isolated In  addition,  example s t a t e d above, c a f f e i n e T. d i g l t a t a . be c o n c l u d e d clear.  A. n o b i l i s .  D.  (58),  (58),  p l a n t n a t u r a l product.  for The  E c h i n o g o r g i a pseudosappo  occurrence  with  of  the f i r s t  and moving through  was  origin  of  of  the  can  caffeine  compounds  from  to i n c o r p o r a t e  microalgae. than  one  not  Chinese  cells  the  in  a  tissues.  o f d o r i d o s i n e (59)  feed  is  the  plant  i n b o t h a sponge and a  on  bacteria  Observation of ageline A  in  nudibranch the  sponge  A case c o u l d be made f o r the  sponge o b t a i n i n g the p u r i n e d e r i v a t i v e from a s y m b i o t i c Sponges  i t can  perhaps be r a t i o n a l i z e d by  g r a z e d upon by the n u d i b r a n c h .  organism.  purine  the compounds o f  d i g e s t i v e system i n d i c a t e s t h a t the compound o r i g i n a t e d which  significant  example, i s a l s o a w e l l known t e r r e s t r i a l  c h a r a c t e r i s t i c a b i l i t y o f gorgonians  The  form  beginning  isolation  symbiotic f a s h i o n into t h e i r  some  s a n d i e g e n s i s and P^. melanobranchia.  t h a t i n most cases the  Caffeine  gorgonian  display  shows t h a t  dietary  micro-  and they c o n t a i n many s y m b i o t i c  (66)  and hokupurine  (73)  in  organism i s f u r t h e r p r o o f o f the d i f f i c u l t y encountered  more i n the  assignment o f o r i g i n f o r these p u r i n e m e t a b o l i t e s . N i t r o c o n t a i n i n g m e t a b o l i t e s are v e r y r a r e from  marine  compounds.  sources. One  /3-nitropropionic  acid  antibiotic  (75),  and  (74),  nitro  isolated  T h i s compound was  activity.  Two  nature,  especially  have been r e p o r t e d to produce a few  o f the f i r s t r e p o r t e d  Asperigillus flavus. of  Fungi  in  new  in  containing 1951  such  compounds  was  by Bush e t a l . ^ from 5  found to have a v e r y  low  level  oxindole a l k a l o i d s , cyclopiamine  i t s enantiomer, c y c l o p i a m i n e B (76)  have  been  isolated  A  from  - 61 -  P e n i c i l l i u m cvcloplum.^3  P. c y c l o p i u m W e s t l i n g  encountered fungus on s t o r e d g r a i n human o r animal  and  cereal  is  a  products  frequently destined  for  consumption.  76 Thaller  at a l . ^  4  i n 1972  r e p o r t e d i s o l a t i o n o f p_-nitrobenzaldehyde  (77)  from the fungus L e p i s t a d i e m i i S i n g e r . The  f i r s t example o f  3,5-dinitroguaiacol  (78)  a  nitro  containing  phenol  appears  r e p o r t e d by Ohta e t a l . ^ 5 i n 1977  to  be  from the r e d  62 alga N a r g i n i s p o r a m a b e r r a n s . isolation  of  To the best of this authors knowledge, the  nitrophenols by Ayer et a l .  example from the marine environment. low  yield  i  n  1984  was  only the second  It i s i n t e r e s t i n g to note that the  3,5-dinitroguaiacol (78),  of  2 9  10 mg from 15 Kg of alga, also  suggests the p o s s i b i l i t y of a microbial o r i g i n . In conclusion, the compounds i s o l a t e d from bryozoans  seem  to  be  of  a dietary or symbiotic  o r i g i n of bryozoan metabolites the  isolation  sponge. of  origin.  Pacific  The dietary  has been suggested by P e t t i t et al.^^a ^  n  the bryostatins from two bryozoans, a tunicate and a  dinoflagellate  collected in hypothesis  toxins  Halocynthia Barkley  as  i i abopa.  Sound,  B.C.  of dietary or symbiotic  t i o n of this hypothesis of  such  the  pectenotoxins.  Similarly,  38 and 39 have recently been i s o l a t e d by Northcote,^6b  tunicate,  family  Northeast  The bryostatins appear to be b i o g e n e t i c a l l y related to a number  nitrophenols a  of  the  nitrophenols  and  a  sponge,  Leucosolenia  lending  further  credence  o r i g i n of these compounds.  could only come through  the  isolation  to  f  r o m  sp., the  Conformaof  the  from some micro-organism, whether i t be phyto-  planktonic, b a c t e r i a l or fungal i n nature.  - 63  INTRODUCTION TO THE  E.  Sponges animals. lacking  (Phylum  They true  -  Porifera) are the most primitive of m u l t i c e l l u l a r  contain  a  relatively  tissue and organs.  simple  except  internal organisation,  A l l members of the phylum are s e s s i l e  and exhibit l i t t l e detectable movement. animals,  SPONGES  Sponges  are  f o r around 150 fresh water species.  primarily  marine  They are found i n  a l l seas wherever there are rocks, s h e l l s , submerged timbers or coral to provide a suitable substratum f o r attachment. Sponges vary greatly i n size.  Certain calcareous species are about  the size of a bean, while others can reach the size of a  square  meter.  Some species are r a d i a l l y symmetrical while the majority exhibit irregul a r , massive, erect, encrusting or branching growth patterns. and  shape  are  generally  influenced  size  by the nature of the substratum,  a v a i l a b i l i t y of space, and v e l o c i t y and water  The  type  of  water  current.  current brings i n oxygen and food and removes waste.  the volume of water estimated to pass through a sponge 10 cm  The  Remarkably, in  height  and 1 cm i n diameter i s about 27.5 L per day. Marine demonstrated size  sponges  feed  on  extremely  small p a r t i c l e s .  I t has been  that the majority of the matter consumed by sponges i s of a  undetectable  by  an  ordinary  microscope,  while  the other food  consumed consists c h i e f l y of bacteria, dinoflagellates and other ton.  Sponges  reproduce  asexually  by budding or by a v a r i e t y of pro-  cesses which involve formation and release of an aggregate of cells  from  the parent sponge.  plank-  essential  In marine sponges, these aggregates are  - 64  c a l l e d gemmules. place  Formation o f these gemmules  i n the f a l l b e f o r e  of winter. enabling  -  the p a r e n t  species  to  exist  i n t e r i o r c e l l s o f the gemmules  great  numbers  sponge d i s i n t e g r a t e s w i t h  These gemmules are a b l e to the  in  withstand  through  emerge  freezing  winter.  ultimately  takes  the  and  onset drying  I n the s p r i n g , developing  into  the an  a d u l t sponge. The  approximately  within four Calcarea,  classes  10,000  based  on  species the  o f known sponges can be  nature  and  carbonate  or  generally  labelled  a d d i n g the a p p r o p r i a t e referring  to  the  by  or  number  four  pronged  encountered i n c a l c a r e o u s yellow,  red or lavender.  sponges ( l e s s t h a n 10 cm  Sponge s p i c u l e s , made up  in identifying  i n s i z e and  sponges.  prefix  to  of  axes)  The  spicules of  types,  the  ending  Class  u s u a l l y separate.  are The  Species  i n height).  monaxins  the or  to  brilliant  They  can  be  found  in  all  the  waters.  " g l a s s sponges", get triaxon  or  their six  A l s o , some o f the s p i c u l e s are o c c a s i o n a l l y f u s e d to form  a l a t t i c e l i k e skeleton b u i l t of name.  (when  o f t h i s c l a s s are the s m a l l e s t o f a l l  name from the f a c t t h a t the s p i c u l e s are always o f the  common  by  colours usually  sponges v a r y from g r e y i s h w h i t e  H e x a c t i n e l l i d a , commonly known as  type.  shape  or - a c t i n e (when r e f e r r i n g to calcarea  of  Spicules  -axons  oceans o f the w o r l d , but are g e n e r a l l y r e s t r i c t e d to s h a l l o w  pointed  Class  the number o f axes or r a y s they p o s s e s s  numerical  number o f r a y s or p o i n t s ) . three  skeleton.  s i l i c o n dioxide deposits, vary  o f t e n s e r v e as u s e f u l c h a r a c t e r s  are  the  comprises a l l members known as c a l c r e o u s sponges, d i s t i n g u i s h e d  by s p i c u l e s composed o f c a l c i u m carbonate. calcium  of  placed  long  This c l a s s elaborates  siliceous  fibers,  hence  their  the most symmetrical sponges, which  65 have  cup,  vase  or urnlike shapes averaging 10 to 30 cm i n height.  contrast to Calcarea, Hexactinellidae are found  at  depths  mainly  deep  water  In  sponges,  of 400 to 950 meters mainly i n t r o p i c a l waters of the  West Indies and the Eastern P a c i f i c from Japan to the East Indies. Class Demospongiae contains the greatest number of species ing  most  of  the  North American sponges.  The majority are marine and  d i s t r i b u t e d from shallow water to great depths. characterized their c e l l s . spongian  by  different  bright  Different  species  are  colours due to pigment granules i n  The skeletons vary, consisting of  fibers  includ-  or a combination of both.  siliceous  spicules  or  Spicules containing species  d i f f e r from those i n Class Hexactinellida i n  that  their  spicules  are  larger manoxins or tetraxons rather than triaxons. Finally,  Class Sclerospongiae  sponges d i f f e r from other sponges i n  having an i n t e r n a l skeleton of s i l i c e o u s spicules and an outer encasement of calcium Sponges,  particularly  and  spongian  carbonate.  those without spicules, often produce large  quantities of i n t e r e s t i n g and b i o l o g i c a l l y active secondary that  are  fouling  thought organisms.  organisms,  the  to  deter Since  the  use  metabolites  p o t e n t i a l predators and i n h i b i t growth of many  sponges  contain  symbiotic  As  evidence  of these compounds for s e l f defence, i t has been observed  that i n the sponge Aplysina f i s t u r l a r i s secondary metabolites be found i n the c e l l s l o c a l i z e d adjacent to the exhalent Pioneering  micro-  true o r i g i n of these compounds i s at times i n question,  however, the majority have been a t t r i b u t e d to the sponges. for  fibers  studies  by  Bergmann ^ 5  can  canals.  only  5 7  on the chemical and ecological  aspects of marine sponges i n the early 1960's i n i t i a t e d intense  studies  - 66 -  leading  to  the  isolation  products e s p e c i a l l y sources  plus  sensitive limited  of  hundreds o f s t r u c t u r a l l y v a r i e d n a t u r a l  s t e r o l s , many o f which were  a few unique t o the marine environment.  analytical  techniques  in  this  period,  Studies  t r a c e and minor  d a t i n g back t o the 1940's and  mainly "conventional"  terrestrial  Due t o a l a c k o f  investigators  sterols,  sterols  poriferasterol carbon  were  neglecting  metabolites. 1960's,  therefore,  yielded  t h a t i s , s t e r o l s which p o s s e s s the normal  19 c a r b o n n u c l e u s p l u s an 8 t o 10 carbon s i d e  29  to  t o t h e study o f o n l y the major components, t h e r e f o r e  the o f t e n more i n t e r e s t i n g  common  common  chain.  Among  the  found i n sponges a t t h i s time were c l i o n a s t e r o l (80) i s o l a t e d o r i g i n a l l y from C l i o n a  sterol, chondrillosterol  81  (81), r e p o r t e d  celata.-*  9  most  (79) and Another  as a major component  - 67 -  of Chondrilla  nucula.^  seemed a t the time  to  be  solely  confined  to  27  and  28 carbon s t e r o l s i n  sponges. Among  the  most  widely  distributed  marine organisms are 2 4 - m e t h y l e n e c h o l e s t e r o l Chalina  arbuscula.^  1  cholesterol  (83)  and  (82), found  originally  c h o l e s t a n o l (84)  found  in as  major components i n numerous sponges.  84 With the advent o f i n c r e a s e d a n a l y t i c a l 1970's  the r e i n v e s t i g a t i o n  of marine sponges has  t i o n and documentation o f s t e r o l s side  technology  possessing  resulted  completely  in  the  early  i n the  isola-  unprecedented  c h a i n a l k y l a t i o n p a t t e r n s and m o d i f i e d t e t r a c y c l i c n u c l e i .  In  the  - 68 -  1970's t o 1980's r e p o r t s o f unusual  sterols  literature.  chains  Remarkable  new  side  began  including  c y c l o p r o p a n e and c y c l o p r o p e n e u n i t s , i n a d d i t i o n l a t e d s i d e c h a i n s were In from  sponge,  cyclopropenyl nature only  Calyx  unit  to  appear those  i n the  containing  extensively  alky-  reported.  1975, F a t t o r u s s o  the  to  e t al.^2 reported niceaensis.  i s o l a t i o n of c a l y s t e r o l (85)  Calysterol  (85)  possessed  a  i n the s i d e c h a i n , a f e a t u r e p r e v i o u s l y observed i n  i n some f a t t y a c i d s .  85 Li  et  al.  0 J  later  reported (86)  propenes, ( 2 3 R ) , 2 3 H - i s o c a l y s t e r o l from C a l y x n i c e a e n s i s . a l o n g w i t h tives,  another  two  nicasterol niceaensis.  (89).  ( 9 0 ) , was r e p o r t e d  A novel  steroidal  steroidal (88)  cyclopropane  cyclopropane and  i n 1985 by P r o u d f o o t e t a l .  6 4  The proposed s t r u c t u r e was c o n f i r m e d by p a r t i a l  These  include  aplysterol  (87)  deriva-  (23S,24S,28R)-  containing  Many s t e r o l s w i t h "extended" s i d e c h a i n s have been sponges.  cyclo-  and (24S), 2 4 H - i s o c a l y s t e r o l  (23R,24R,28S),23,24-dIhydrocalysterol  23,24-dihydrocalysterol  two  sterol,  from Calyx synthesis.  encountered  (91),stelliferasterol  in  (92) and  - 69 -  isostelliferasterol  (93), which a l l are  a d d i t i o n o f an e x t r a carbon a t  Three gulasterol and  in  characterized  a  unusual  s i d e c h a i n s are found i n veron-  (94), a minor s t e r o l from the sponge (95),  the  C-26.  f u r t h e r examples o f extended  xestosterol  by  Verongia  cauliformis.^6  major component, and x e s t o s t a n o l  (96), a  70 minor component of Xestosponpia muta. ' These three sterols possess the  R  unique feature of being alkylated at both the C-26 and C-27 positions. Recent  isotope feeding studies have led to the demonstration of de (95)  novo biosynthesis of xestosterol nonstereoselective 20).  from  desmosterol  viaa  SAM biomethylation and a 1,2 hydrogen s h i f t (Scheme  6 8  Modifications to the s t e r o l nucleus are also well sponges.  A  mixture  of stanols  having  a  documented  been  component,  isolated fully  from  ergost-trans-22-en-3;9-ol structures  (99)-(105).  side  the sponge A x i n e l l a polypoides.^9  characterized by spectral (98),  has been  from  19-norcholestanol nucleus  carrying either saturated or unsaturated 8, 9 or 10 carbon has  (97),  data  found  chains  The major  as 19-nor-5a,10/?along  with  related  Scheme 20:  9 9R  - H  100  - Me  R  Biosynthesis  -|04  of xestosterol  R - Et, A - t r a n s 22  (95)  105  72 -  E x a m i n a t i o n o f the isolation marine  of  first  environment.  sterols,  (106)-(111),  H0H C 2  In  the  1982,  sponge A x i n e l l a v e r r u c o s a example  A s many as  7 0  o f an A - n o r c h o l e s t a n e six  side  chains,  H  et  a l .  methyl-A-nor-5a-cholest-15-ene  7  1  nucleus  to  the  from  the  3/?-hydroxymethyl-A-nor-5a-cholestane  containing various  Eggersdorfer  i n 1974 l e d  reported  (112)  112  the  from  were  isolated.  106  R - H  107  R - Me  108  R - Et  109  R - H,  A  110  R - Me,  A22  111  R - Et,  A22  2 2  i s o l a t i o n o f 3/S-hydroxythe  pacific  sponge,  - 73 -  Homaxinella t r a c h y s . s t e r o l w i t h a C-15, Biosynthetic et  al.'^  suggest  This C-16  represents  the  first  naturally  double bond.  t r a c e r i n c o r p o r a t i o n s t u d i e s c a r r i e d out  by  Bibolino  the A-nor s t e r o l s can be formed de novo from r e a d i l y  a v a i l a b l e c h o l e s t e r o l v i a an enzyme c a t a l y z e d  r i n g c o n t r a c t i o n mechanism  i n v o l v i n g the l o s s o f the 3a and 4/3 hydrogens and the f o r m a t i o n l i n k a g e between C-2 and C-4  Scheme 21:  occurring  o f a C-C  (Scheme 21).  Biosynthetic conversion of cholesterol into methyl-A-nor-cholestane  3/3-hydroxy-  -  74  This i s the most convincing evidence sponges  to  so  far  for  the  ability  of  chemically modify sterols subsequent to dietary intake.  It  has also been found that sponges which contain these A-nor sterols  lack  7 ^  sterols  with  conventional  nuclei.  This  possibly  indicates  e f f i c i e n c y of the enzymic system i n converting absorbed dietary into  A-nor s t e r o l s .  in  some  sterols  Therefore, the existence of as yet unknown sterols  in nature with conventional detected  the  cases  nuclei through  but  unusual  side  chains  can  be  i s o l a t i o n of the corresponding A-nor  sterol. The most common theories on the d i v e r s i t y of marine sterols to  be  due  to the accumulation  appear  of several phenomena, including genetic  control, biosynthesis by zooxanthellae, commensalism and parasitism, and transmission  of  molecules  by  the food chain or by sea water.  biochemical standpoint, structural v a r i a t i o n i n side  chains v i a b i o l o g i c a l methylations  modification  of  sterols  is  form  of  extended  has been shown to modulate c e l l  p l a s t i c i t y , membrane permeability and ion nuclear  the  From a  as  exchange. yet  74  The  effect  of  unclear, however, i t is  believed to also play a specialized role i n membrane function.  - 75 F.  NOVEL STEROIDS FROM THE SPONGE Anthoarcuata graceae (Bakus  1.  1966)  Introduction  Anthoarcuata graceae  (Bakus  1966)  is  a  reddish  (family Plocamiidae, order P o c c i l o s c l i d a , class Homoscleromorpha) habitats of the cooperi  is  commonly  B.C.  often  found  coast.  found  in  The  rocky  dorid  orange  Demospongiae, intertidal  nudibranch  sponge subclass  and  subtidal  A l d i s a sanguinea  i n close association with the sponge. -  5  Most  A^ graceae  from  7  often the nudibranch i s deeply embedded i n the  sponge  which i t obtains n u t r i t i o n and pigments giving i t c r y p t i c coloration. Our  chemical  i n v e s t i g a t i o n of yielded  a  studies the  mixture  g l y c e r o l ether 115  on  Aj. graceae  chemistry of  of  the  biologically  were  nudibranch  active  and a complex mixture  prompted  of  a prior  A^. cooperi  steroids minor  by  113  which  and 114, a  steroidal  ketones,  fats and s t e r o l s which were not studied further. -* 7  A. graceae.  c o l l e c t e d i n the shallow waters of Barkley Sound, B.C.  at various times of the year was metabolites  found  in  to  be  lacking  in  the  major  A^. cooperi. however, a preliminary investigation  did reveal the presence of several steroids.  found  other  minor  but  very  interesting  - 76 -  115 2.  I s o l a t i o n and Structure Elucidation  Anthoarcuata praceae was c o l l e c t e d depth)  and  immediately  immersed  in  by  hand  methanol  using (or  SCUBA  ethanol).  extraction f o r one to three days at room temperature, the ethanol)  m  After  methanol  (or  was decanted, vacuum f i l t e r e d and evaporated i n vacuo to y i e l d  an aqueous between  (1-5  methanolic  brine  and  anhydrous Na2S0^.  suspension.  chloroform,  and  This the  suspension  was  partitioned  organic layer was dried over  The sponge was further soaked i n  dichloromethane  at  room temperature for one day, before being ground (with dichloromethane) i n a Waring blender. was  The suspension of ground sponge i n dichloromethane  vacuum f i l t e r e d through c e l i t e to y i e l d a f i l t r a t e which was  stored  - 77 at below room temperature. with  dichloromethane  a  The r e s u l t i n g sponge solids  chloroform,  i n vacuo, p a r t i t i o n e d  and the organic layer was  F i l t r a t i o n and evaporation i n afforded  extracted  further three times at one day i n t e r v a l s .  combined f i l t r a t e s were evaporated and  were  vacuo  of  a dark red crude o i l which was  between  dried over anhydrous  the  combined  analytical  TLC  analysis.  Further  purification  mixture  of  NMR  c a r r i e d out  A -3,6-diketosteroids 4  A (118)  116  phase  Analysis of these  by and  a  preparative 117,  and anthosterone  two B (119)  TLC  yielding  two  novel  unprecedented A-nor-steroids as well as two  diosphenols  121.  A -3,6-diketosteroids 4  Compound  A,  mp 92-93°C, had a molecular formula of  396.3034, calcd. 396.3030) that required 8 units of *H NMR  methyls  C27H40O2  (HRMS  unsaturation.  The  indicated methyl resonances at 6 0 . 7 3 (s, 3 H ) , 0 . 9 5 (d, J - 6 Hz,  3 H ) , and 1.17 ppm  NMR  reverse  Further separation and p u r i f i c a t i o n of  t e n t a t i v e l y assigned structures 120 and  3A.  detected  provided evidence f o r the presence of  components i n each.  each mixture was  anthosterone  C  layers  of the s t e r o i d a l  and  LH-20)  HPLC y i e l d e d two s t e r o i d a l fractions as white s o l i d s . 1 3  Na2S0^.  fractionated by f l a s h chromato-  f r a c t i o n by column chromatography (Sephadex  two fractions by ^H and  brine  organic  graphy to give complex mixtures of f a t s , steroids and pigments by  The  (s, 3 H ) , corresponding to the  of a conventional steroid.  resonances were found at 6 1.61  C-18,  C-21,  and  C-19  In addition, two o l e f i n i c methyl ^H (s, 3 H ) , 1.69  (s, 3 H ) , as well as an  78 -  120  - 79 olefinic  triplet  presence of a A supported  by  2 4  at 5.09 ( t , J - 6.4 Hz, IH), ppm, which indicated the •  double bond i n the side chain.  2 5  an IR absorbance at 1643 cm"  This  was  further  and C NMR resonances  1  1 3  at 6  124.99 (d), 127.09 (s) ppm which could be assigned to the C-24 and  C-25  carbons of the side chain. One  remaining  o l e f i n i c signal i n the ^H NMR at S 6.19 (s, IH) ppm  (Figure 11), i n addition to four downfield resonances indicated rings.  the  The  1 3  presence  of  C NMR resonances  some  unique  i n the  1 3  C  NMR,  f u n c t i o n a l i t y i n the A and B  at 5 202.36 (s) and 199.56 (s) ppm  were  reminiscent of the carbonyl carbon resonances  found i n A -3-ketosteroids  (Table 8), while the remaining two resonances  at 8 125.48 (d) and 161.06  4  (s) ppm could be assigned to the corresponding A -double bond. 4  Infrared  bands at 1606 (vc - c) amd 1686 (i/c - ojcm" , coupled with a UV A 1  248.9  nm  (e  11600)  representing  m a x  at  an enone with extended conjugation,  allowed the tentative assignment of compound A 116 as an ene-dione. Confirmation of the assigned structure was achieved by sis  of  a  Scheme 22:  model  A -3,6-diketosteroid 4  system  Jones o x i d a t i o n o f c h o l e s t e r o l (83)  (Scheme  the synthe-  22). This was  Figure 12:  400 MHz H NMR of compound 122 X  - 83 Table 7:  H on C  #  1  H NMR Data f o r A -3,6-Ketosteroids H  (116)  (CDCI3)  Chemical S h i f t , 5, ppm (117)  a  a  (122)  a  4  6 19 (s,lH)  6 17 (s.lH)  6.19 (s, IH)  18  73 (s,3H)  73 (s,3H)  .74 (s, 3H)  19  1 17 (s,3H)  1 17 (s,3H)  1.19 (s, 3H)  21  95 (d,J=6 Hz,3H)  24  5 09 (t,J°6.4 Hz.lH)  26  1 69 (s,3H)  b  27  1 .61 (s,3H)  b  28  85 (d,J=7 Hz,3H)  .94 (d, J=6 Hz, 3H)  1 02 (d,J=8 Hz,3H)  .87 (d, J=6 Hz, 3H)  -  -  4 .65/4.73  400 MHz s h i f t s interchangeable  116  11 7  0  Figure 14:  400 MHz H NMR of compound 117 i  - 86 Table 8:  i J  H on C  #  C  NMR  Data f o r A -3,6-diketosteroids 4  (CDCI3)  Chemical S h i f t , 6, (117)  (116)  ppm (122)  3  199.,56 (s)  199.,56 (s)  199.,40 (s) ,  4  125..48 (d)  125,.44 (d)  125..33 (d)  5  161.,06 (s)  161.,08 (s)  160,,98 (s)  6  202,.36 (s)  202.,36 (s)  202.,25 (s)  24  124.,99 (d)  156..65 (s)  25  127,,09 (s)  28  -  106,.06 (t)  122  - 87  Scheme 23:  -  MS f r a g m e n t a t i o n o f compound B 117  - 88 achieved v i a a purification  Jones  by  oxidation ^ 7  preparative  one of the major products. ponding  to a  molecular  of cholesterol  (83) which  TLC gave A -cholestane-3,6-dione (122) as 4  The HRMS of 122 gave a formula  of C27 42^2 H  parent  A H NMR spectrum of 122 (Table 7) showed a  signal  5  1  6.19  (s, IH) ppm  ion corres-  (HRMS 398.3186, calcd.  398.3187).  at  after  sharp  olefinic  (Figure 12) and i t s C NMR spectrum 1 3  (Figure 13) had resonances at S 199.40 (s) and 202.25 (s) corresponding to  the carbonyl  carbons  (C-3, C-6) plus two o l e f i n i c resonances at 6  125.33 (d) and 160.98 (s) ppm (C-4, C-5) assigned bond  system  (Table  7).  to  the A • 4  5  double  The IR spectrum of 122 showed absorbances at  1685 and 1606 cm" i n close agreement to the natural product. 1  Co-occurring with Compound A 116 was a related steroid, Compound 117,  mp  111-112°C,  which  possessed  a A  24,2  B  ** double bond i n the side  chain.  Compound B 117 had a molecular formula C 2 8 H 4 2 O 2 (HRMS  410.3190,  calcd.  410.3187) d i f f e r i n g from compound A 116 by 14 mass units  (=CH2).  The H NMR (Figure 14) showed methyl resonances at 6 0.73 (s, 3H), 0.85 l  (d,  J = 7 Hz, 3H), 1.02 (d, J - 8 Hz, 6H), 1.17 (s, 3H) ppm correspond-  ing to the C-18, C-21, C-26/27 and C-19 steroidal  methyls  respectively,  of the  skeleton (Table 7). ^H NMR o l e f i n i c resonances at 6 4.65 (s,  IH) and 4.73 (s, IH) ppm, i n addition to C NMR (Figure 15) signals 1 3  S  106.06  (t) and 156.65 (s) ppm, are t y p i c a l of an olefinic-methylene  at the C-24/28 p o s i t i o n (Table 8). This i s further strong ^6^12  slc  at  peak  at m/z  by the  326 i n the mass spectrum indicating a loss of the  *e chain v i a a McLafferty rearrangement  functionality  supported  (Scheme 23) .  Identical  i n the A and B rings was confirmed by the presence of a  sharp o l e f i n i c s i n g l e t at 5 6.17 (s, IH) ppm  i n the ^H  NMR,  and b y  - 89 resonances  at S 125.44 (d), 161.08 (s) (A ' -double bond), 199.56 (s), 4  5  202.36 (s) ppm (carbonyl carbons) i n the C NMR.  Compounds A and B had  1 3  comparable UV and IR spectral data.  3B.  A-Nor Steroids  Examination  of the more polar band from the preparative TLC puri-  f i c a t i o n y i e l d e d two related compounds anthosterone A (118) and terone  B  (119).  Anthosterone  A (118), mp 142-143°C, had a molecular  formula C 8H42°4 (HRMS 442.3083, calcd. 442.3079) requiring 8 2  unsaturation.  Its  NMR  anthos-  units  of  spectrum gave methyl signals at 8 0.73 (s,  3H), 0.96 (d, J = 6.7 Hz, 3H), and 1.21 (s, 3H) ppm corresponding to the C-18,  C-21  and C-19 methyls of a s t e r o i d skeleton, i n addition to two  signals assigned to o l e f i n i c methyl groups (C-26/C27) at 8 1.62 (s, 3H), and  1.69  (s, 3H) ppm.  Further  examination  of the ^H NMR spectrum  (Figure 16) indicated the presence of an o l e f i n i c t r i p l e t at 8 5.10 ( t , J A  -  8  Hz,  IH) ppm  which could be assigned to a proton on C-24 of a  24,25 double bond i n the side  chain  (Table  9).  1 3  C  NMR  olefinic  resonances at 125.09 (d) and 131.05 (s) supported this assignment (Table 10).  Additional ^H NMR resonances consisted of an AB quartet made up of  doublets resonating at 8 2.08 (d, J - 13.3 Hz, IH) and 2.19 (d, J - 13.3 Hz, IH), one sharp methyl singlet at 3.79  (s, 3H), and a deshielded  o l e f i n i c proton at 6.73 (t, J = 3.3 Hz, IH) ppm.  90 -  - 91 -  92  -  - 93a-  -93 b -  94 -  T a b l e 9:  • H NMR L  data f o r compounds 118 and 119  Chemical H on C  #  118  shift,  6 ppm  119  a  a  1  2.08/2.19 (d, J-13.3 Hz, IH)  2.10/2.18 (d, J-13.5 Hz, IH)  5  6 73 ( t , J=3.3 Hz, IH)  6.73 ( t , J=2 5 Hz, IH)  18  0 73 ( s , 3H)  0.73 ( s , 3H)  19  1 21 ( s , 3H)  1.20 ( s , 3H)  21  0 96 (d, J=6.7 Hz, 3H)  0.97 (d, J=6 5 Hz, 3H)  24  5 10 ( t , J=8 Hz, IH)  26  1 62  b  (s . 3H)  27  1 69  b  (s , 3H)  28  3 79 ( s , 3H)  1.04 (d, J-6 5 Hz, 6H)  29  4.65-4.71 (s 3.77 ( s , 3H)  11 8  119  IH)  - 95 Table 10:  i J  C NMR data f o r compounds 118 and 119  Chemical s h i f t , 5 ppm H on C  118  #  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29  t a  56 79 173 200 145 136 32 32 49 42 22 39 39 56 24 28 55 12 21 35 18 24 36 125 131 17 25 53  02 90 37 91 10 28 21 32 66 94 11 92 35 26 35 11 91 03 79 55 66 35 06 09 05 66 75 39  1  (t) (s) (s) (s) (s) (d) (t) (d) (d) (s) (t) (t) (s) (d) (t) (t) (d) (q) (q) (d) (q) (t) (t) (d) (s) (q) (q) (q)  Assignment based on APT experiment (Figure 20). 75 MHz, CDC1  3  k  Assignments interchangeable  c  Assignment interchangeable  H9T  46 68 79 89 173 36 200 91 145 08 136 29 32 21 32 32 49 64 42 95 22 01 39 93 39 34 56 25 24 34 28 13 55 93 12 03 22 11 35 72 18 74 30 99 34 64 153 53 33 81 21 87 21 79 106 01 53. 40  a  (t) (s) (s) (s) (s) (d) (t) (d) (d) (s) (t) (t) (s) (d) (t) (t) (d) (q) (q) (d) (q) (t) (s) (s) (q) (q) (t) (q)  v  b  c c  96 Assignment  of  the sharp methyl singlet at 5 3.79 was aided by the  observation of a MS fragment at m/z 383.2932 (calcd. 383.2952) ing  an  M -59  loss corresponding to  +  C2H3O2 of a methyl ester function-  ality.  The presence of the methyl ester moiety  by  NMR  1 3  CH3)  C  indicat-  was  further  confirmed  resonances at 6 173.37 (s) (C-4, C-0) and 55.91 (q) (C-29,  ppm (Figure 17) along with an IR band at 1744 cm" . 1  An i n t e r e s t i n g absorbance i n the IR spectrum at 1717 cm" , 1  coupled  with a C NMR carbonyl signal at 6 200.91 (s), two o l e f i n i c signals at 1 3  6 136.28 (d) and 145.10 (s) ppm, and a UV A  m a x  at 250.2  (c  12500)  nm  suggested the presence of an enone system as seen i n compounds A and B. The  mass spectrum of 118 also showed a strong peak at m/z 330.1810  ( 20 26°4> calcd. 330.1832) i n d i c a t i n g a M -113 ( C H ) loss correspondC  H  +  g  ing  to  the cleavage  (calcd.  of the side chain (Scheme 24). With the further  (C2H4O2) as indicated  loss of 60 daltons 270.1621),  of a  by  a  peak  at m/z  270.1597  the remaining nucleus after the loss of the methyl  ester yielded a molecular formula C ^ g H 2 2 0 presence  17  nor-sterol  (Scheme  which  2  24).  With  first  indicated  the  a l l the methyl groups  accounted f o r , a l o g i c a l conclusion to draw was the presence of an A-nor ring. Co-occurring  with  anthosterone B (119), C  29 44°4  (HRMS  H  ration. 0.97  anthosterone mp  155-157°C,  A  (118) was the related steroid,  which  had  a  molecular  formula  456.3236, calcd. 456.3241) requiring 8 units of unsatu-  The H NMR (Figure 18) showed methyl signals at 6 0.73 (s, 3H),  (d, J  1  =  6.5 Hz, 3H), 1.04 (d, J = 6.5 Hz, 6H), 1.20 (s, 3H) ppm  assigned to the C-18, C-21, C-26/27 and C-19 difference  i n molecular  weight  of  14  methyls  daltons  respectively.  A  ( C H 2 ) plus o l e f i n i c  - 97 Scheme 24:  MS fragmentation of anthosterone A (118)  m/z 383 (10 V.)  resonances i n the H 1  NMR  at  6  4.65  (s, IH), and  4.71  (s, IH)  ppm indicated the presence of a C-24 (28) olefinic-methylene  system (Table  9).  at  This assignment was supported by the C NMR resonances 1 3  106.01  (t) and 156.79 (s) ppm (Figure 19) which are diagnostic f o r this type of system. presence  The remaining portions of the ^H of  the AB  quartet  NMR  spectrum  (2.10/2.18, d, J - 13.3 Hz, 2H), a sharp  s i n g l e t a t 6 3.77 (s, 3H) f o r the methyl ester as well triplet  at  including the  as  an  olefinic  6.73 (t, J - 3 Hz, IH) ppm indicated that the basic nucleus  - 98 of anthosterones A and B was i d e n t i c a l . NMR  spectrum  which  This was supported by  the  ^-^C  showed a deshielded carbonyl resonance at <5 200.91  (s), two o l e f i n i c resonances at 136.29 (d) and 146.08 (s) ppm (Table 10) and  the mass  spectrum  which  also  showed  a strong peak at 298.1928  (calcd. 298.1934) corresponding to the C20H26O2 nucleus (Scheme 2 5 ) .  Scheme 25:  MS fragmentation of anthosterone B (119)  119 m/z 456 ( 2 •/•)  m/z 382(29'/.)  m/z 298 (48 V.)  Since the difference between the two compounds was  only  i n their  chains, the remaining objective was to propose a structure for t h e  side  common nucleus. Further examination of the showed  the presence  NMR spectrum of anthosterone A (118)  of a carbon singlet at 6 79.90 (s) assigned t o a  - 99 -  t e r t i a r y carbinol carbon. ported  The presence of a  hydroxyl  was  sup-  by the mass spectral loss of HOH (Scheme 24). As expected then,  anthosterone B (119) could be acetylated with acetic at  group  room  temperature  anhydride/pyridine  f o r 12 hr to afford anthosterone B-acetate (123)  498.3336 (calcd. 498.3347) i n quantitative y i e l d (Scheme 2 6 ) .  Scheme 26:  7 7  Acetylation of anthosterone B (119)  123 The !H NMR spectrum of 123 (Figure 21) showed an u p f i e l d  shift  in  the C-19 methyl resonance from 5 1.20 i n 119 to 1.10 ppm i n 123, as well as a downfield s h i f t i n both protons making up the AB system, doublets  at  5  2.10  and  2.18  i n 119  with  the  s h i f t i n g to 2.47 and 2.65 ppm  respectively i n 123 (Table 11). This indicated a close proximity i n the  - 100 -  101 Table 11:  *H NMR comparison between 119 and 123  O  0  123  Chemical s h i f t , 5 ppm H on C  119'  #  123'  2.10 (d, J-13.5 Hz, IH)  2.47 (d, J=14 Hz, IH)  2.18 (d, J=13.5 Hz, IH)  2.65 (d, J - 15 Hz, IH)  18  0.73 (s, 3H)  0.72 (s, 3H)  19  1.20 (s, 3H)  1.10 (s, 3H)  21  0.97 (d, J-6.5Hz, 3H)  0.97 (d, J=8 Hz, 3H)  1.04  1.03 (d, J-8 Hz, 6H)  26 27 28  4.65/4.71  (s, IH)  4.67-4.73  (s, IH)  -  -  102  nucleus between the hydroxyl group, the C-19 system.  A  methyl  and  the  AB  spin  difference NOE experiment, i n which the C-19 methyl singlet  of anthosterone B (119) was irradiated, showed  an  enhancement  of  the  u p f i e l d portion of the AB quartet showing the close spacial proximity of these f u n c t i o n a l i t i e s . of  the  olefinic  Additional NOE experiments included  t r i p l e t at 6" 6.73 ppm which induced an enhancement of  a l l y l i c proton multiplets at 6 1.88 irradiation  irradiation  (m, IH), and 2.41  (m,  IH)  of the multiplet at 6 2.41 (m, IH) y i e l d i n g an NOE  ppm  and  enhance-  ment of the t r i p l e t at S 6.73 as well as i t s apparent geminal partner at 1.88  (m,  IH)  ppm.  I r r a d i a t i o n of the o l e f i n i c signal at 6 6.73  double resonance experiment resulted i n sharpening of and  2.40  ppm, while i r r a d i a t i o n of the multiplet at S 2 AO  t r i p l e t at 6.73 1 3  C  NMR  at  spectrum  1.88  changed the  to a doublet and sharpened the signal at 1.88 ppm.  The  showed two o l e f i n i c signals, 6 136.28 (d) and 145.10  (s) ppm, which could be assigned to a adjacent a l l y l i c methylene (Figure 22).  Figure 22:  signals  in a  A l l y l i c methylene system  t r i - substituted  alkene  with  an  - 103 The next major obstacle was the positioning of the ketone  carbonyl  from the UV data had to be conjugated to a double bond.  Assuming  which  the presence of the contracted structures  based  on  A  ring,  proposed  possible  to  propose  a l l the accumulated data which possessed either a  4-keto or a 6-keto f u n c t i o n a l i t y . were  i t was  Three possible structures A, B, and C  which accommodated a l l the f u n c t i o n a l i t y required by the  data, including the methyl ester, enone system, t e r t i a r y alcohol as well as the f i v e membered A r i n g .  G A  SINEPT ^ 7  (selected  transfer) experiment observed  i n s e n s i t i v e n u c l e i enhanced by p o l a r i z a t i o n  eliminated  one  of  the  possibilities  p o l a r i z a t i o n transfer through 2 and 3 bond  - C 1 3  based  couplings.  A s o f t pulse applied to the u p f i e l d proton of the AB system (5 2.18 afforded  enhancements  of  1 3  C  NMR  on  ppm)  signals at 200.91 ( s ) , 173.36 (s),  - 104 145.08 (s) and 79.90 (s) ppm indicating ^-H - C  coupling  1 3  unsaturated  ketone  carbonyl,  the methyl  ester  protonated o l e f i n i c and the carbinol carbons tures  A  and  C  to  carbonyl,  (Figure 23).  the a,/3 the non-  Only  struc-  could f i t this data while with structure B, a required  6-bond coupling to  the methyl  ester  carbonyl  carbon  would  not be  expected. A  two dimensional homonuclear COSY  79  experiment  AB system was not spin i s o l a t e d as indicated between  the  downfield  member  by  an  confirmed that the observed  coupling  of the AB system at 6 2.18 and the C-19  methyl s i n g l e t at 1.20 ppm (Figure 24). This observed coupling (W-coupling)  between  the methyl  group  and  the downfield a-proton provided  strong evidence f o r the proposed structure C. istry  at  the  stereochem-  C-2 center with the hydroxyl group i n the 0 orientation,  was assigned based on the observed u p f i e l d resonance  The r e l a t i v e  shift  of  the  C-19  methyl  (1.20 to 1.10 ppm) on acetylation of anthosterone B (119).  Confirmation  of  the  X-ray d i f f r a c t i o n a n a l y s i s  structure was 8 0  provided by a single c r y s t a l  on anthosterone A (118).  computer generated ORTEP drawing of 118.  Figure 25 shows a  IRRAD. AT 2.18 ppm  200.91  Figure 23:  SINEPT experiment on anthosterone B 119  - 106 Figure 24:  2D Homonuclear COSY on anthosterone B (119)  ^2 0  ( "V) P  4 -|  3.2  2 8  2.4  2 0  1.6  Fl  Figure 25:  1.2  0.6  0  4  (PPM)  Computer generated ORTEP drawing of anthosterone A 118  - 107 3C.  Diosphenols 120 and 121  An  investigation  of  two  steroids which were more polar than the  A-nor s t e r o l s on preparative TLC, and present i n trace amounts, has to a preliminary s t r u c t u r a l proposal based on spectral data. 120, had a molecular formula C27H42O3 (HRMS requiring  7  units  signals at S 0.73 assigned  to  of  unsaturation.  (s, 3H), 0.93  the C-18,  The  414.3149, NMR  C-21 and C-19 methyls of a s t e r o i d nucleus.  Two  and anthosterone A (118).  (s, 3H) and 1.69  ( t , J - 7 Hz, IH) ppm  (s, 3H)  in  compound  (d, J = 16 Hz, IH) and 2.25  at 3.29  plus  indicated the presence  Further examination of the ^H NMR  revealed an AB system, 6 2.18 singlet  spectrum gave methyl ppm  of a t r i s u b s t i t u t e d side chain double bond as seen  sharp  414.3136)  (s, 3H)  an o l e f i n i c t r i p l e t at 5.09  a  Compound E  calcd  (d, J -= 6 Hz, 3H), and 1.28  o l e f i n i c methyl groups (C26/27) at 6 1.52  IH),  led  A  116  (Figure 26)  (d, J = 16 Hz,  (s, 3H), a deshielded methine proton at  Figure 26:  400 MHz H NMR of compound E 120 L  - 109 4.52  (t, J  = 3 Hz, IH) as well as an o l e f i n i c singlet at 5.28 (s, IH)  ppm (Table 12). (MeOH),  Based on a loss i n the mass  spectrum  of 32  daltons  the three proton singlet at 3.29 was assigned to a methyl ether  f u n c t i o n a l i t y (Scheme 27).  Infrared bands at 3438 (i>0-H) , 1711, 1674 (wC=0) and cm" , 1  9731) phenol  plus  1606  (i/C=C)  a UV absorbance 264 nm (e 10971) which s h i f t e d to 304 nm (e  on addition of base, strongly indicated the presence functionality  based  on  of a  dios-  a comparison with l i t e r a t u r e v a l u e s . ^  The presence of the diosphenol was further supported by the positive TLC spray test with 2,4-dinitrophenylhydrazine^  2  and F e C ^ . ^  3  110 T a b l e 12:  X  H NMR  data on d i o s p h e n o l s 120 and 121  Chemical s h i f t , H on C  120  #  a  2.18/2.25 (d, J-16 Hz, IH)  1  6 ppm 121  a  2.17/2. 29 (d, J-16 Hz, IH)  4  5.28 ( s , IH)  5.28 ( s , IH)  6  4.52 ( t , J-3 Hz, IH)  4.57 ( t , J-2 Hz, IH)  18  0.73 ( s , 3H)  0.74 ( s . 3H)  19  1.28 ( s , 3H)  1.31  21  0.93 (d, J - 6 Hz, 3H)  0.94 (d, J -  23  5.09 ( s , IH)  25  1.52  b  ( s , 3H)  1  26  1.69  b  ( s , 3H)  J  3.29  -  28  400 MHz, shifts  CDC1  4.76 ( s , IH) 3.33 ( s , 3H)  interchangeable  120  3H)  1.03 (d, J - 7 Hz, 6H)  3  OCH3  8 Hz,  \  27  ( s , 3H)  OCH3  121  - Ill I r r a d i a t i o n of the C-19 methyl singlet at 6 1.28 NOE  experiment  on  compound  E  i n a difference  120, resulted i n an enhancement of the  methoxy s i n g l e t at 3.29 and the u p f i e l d component of the AB 2.18  (d, J = 16 Hz, IH) ppm.  at  8  Decoupling of the  4.52 sharpened a l l y l i c signals at 2.10 as well as proton  signals i n the a l i p h a t i c region at 1.00 to 1.20 ppm. homonuclear  at  I r r a d i a t i o n of the t r i p l e t at 5 4.52 ppm  afforded only an enhancement of the methoxy s i n g l e t . triplet  system  COSY  experiment  on  A two  dimensional  compound E further suggested coupling  between the proton at <S 4.52 and an a l l y l i c proton at 2.10 and a l i p h a t i c protons at 1.00 to 1.20 ppm (Figure 27). Co-occurring which had  a  428.3291).  with  molecular A  1  H  compound formula  E 120 was the related compound F 121, of  C28H44O3  (HRMS  428.3284,  calcd.  NMR of compound F 121 afforded signals at 6 0.74 (s,  3H), 0.94 (d, J - 7 Hz, 3H), 1.03 (d, J - 6.4 Hz, 3H), and 1.31 (s, 3H) ppm  assigned  The presence 4.69  to the C-18, C-21, C-26/27 and C-19 methyls respectively. at 8  i n the ^H NMR (Figure 28) of sharp o l e f i n i c s i n g l e t s  (s, IH) and 4.76 (s, IH) ppm (Table 12) plus an additional 14 mass  units ("CH2) i n the mass spectrum indicated the presence of an o l e f i n i c methylene  functionality  i n the side chain as seen i n compounds 117 and  119. Subtracting the two  carbons  assigned  to  the methyl  ether  o l e f i n i c methylene from the t o t a l of 28 carbons i n the molecular of compound F, leaves a compound  total  of  only  26  carbon  atoms.  F cannot have a standard s t e r o i d a l skeleton.  t i o n of the mass spectrum of compound F 121 revealed a m/z  344.2351  and  formula  Therefore  Further examinastrong  peak at  (calcd. 344.2353) i n d i c a t i n g a loss of 84 daltons ( C H ) 6  12  112 -  114 assigned to loss of a truncated side chain v i a a ment  (Scheme  28).  A  similar  McLafferty  rearrange-  fragmentation pattern was observed f o r  compound E.  Scheme 28:  MS fragmentation of compound F 121  m/z A28 (5 7.) m/z  This evidence allowed the assignment of shortened  3 U (12 •/.)  side  chains  in  both compounds E and F. Based on the spectroscopic data accumulated  i n these two compounds,  tentative structures 120 and 121 were assigned pending further i s o l a t i o n of  s u f f i c i e n t material which would f a c i l i t a t e an unambiguous assignment  of t h e i r structures.  115  3D.  Biosynthesis of Sponge Metabolites  A  biosynthetic  isolated  from  proposal  Anthoarcuata  A -cholesterol.  A  24  A -3,6-diketosteroids 4  chain  found  for graceae  proposed  Allylic  formation could  oxidation  logically  biogenetic  pathway  rearrangement  of  at  the  begin  29.  the  double  with  the The  two side  biomethylation  and a l l y l i c rearrangement (steps  2  and  bond (step 4) followed by and  allylic  hydroxylation  C-6 p o s i t i o n (steps 6, 7) represents a possible  biogenetic route to compounds 116 and A mixture of  steroids  for  i n compounds 117 and 119 could be formed by  oxidation of the 3^-hydroxyl group (step 5) and  of the new  116 and 117 i s outlined i n Scheme  with S-adenosylmethionine (SAM) 3).  the  steroids  117.  including  4,7,22-triene-3,6-diketones  124  have been i s o l a t e d by Malorni et a l . ^ from the marine sponge, Raphidos4  t i l a incisa.  collected  off  diene-3,6-dione (125), was  Zlarin,  Yugoslavia,  and  cholesta-4,7-  isolated by Kinnear et al.^- from the i n s e c t , >  R  0  124  R=H  125  R=H  2  116 Scheme 29:  Biosynthetic proposal  f o r compounds A and B  - 117  Calliphora stygia. the  only  -  I t appears, however, t h a t compounds 116  examples  of  naturally  and 117  are  occurring A -3,6-diketosteroids  from  4  marine s o u r c e s . A p o s s i b l e b i o s y n t h e t i c pathway t o the a n t h o s t e r o n e s (119)  proceeds  through  the  a-diketone  tautomer  o f the A r i n g can o c c u r v i a a b e n z y l i c a c i d  (Step 2) t o g i v e the a-hydroxy c a r b o x y l i c a c i d hydroxylation  and  (118)  and  B  a d i o s p h e n o l i n t e r m e d i a t e as seen i n Scheme 30.  I f the d i o s p h e n o l i s c o n v e r t e d t o contraction  A  (Step  1),  rearrangement ^ 8  intermediate,  which  on  o x i d a t i o n (Step 3), f o l l o w e d by m e t h y l a t i o n (Step  would form the common n u c l e u s found i n n a t u r a l p r o d u c t s  and 119.  It  appears  from a l i t e r a t u r e s e a r c h t h a t the n u c l e u s found i n compounds  118  and 119  i s unprecedented.  unusual  A-nor  steroids  I t i s not c o m p l e t e l y c l e a r y e t whether are  metabolites  a r t i f a c t s formed d u r i n g i s o l a t i o n . an  attempt  elaborated  A proposed  pathway t o the d i o s p h e n o l s 120  SAM,  T h i s experiment  and 121  s i d e c h a i n s s h o r t e n e d by one carbon are known from previous  examined  is  was  outlined  marine  organisms.  b i o s y n t h e t i c p r o p o s a l i n v o l v e d the m e t h y l a t i o n o f C-24  32).  8 8  F  chains  121  proposed  for  compounds  120  and  (Scheme appear  n a t u r a l l y o c c u r r i n g examples o f these s i d e c h a i n s .  32).  87  with chain  Subsequent m e t h y l a t i o n s and r e d u c t i o n s c o u l d y i e l d  s i d e c h a i n s found i n the d i o s p h e n o l s E and  in  p o s i t i o n . Examples o f  f o l l o w e d by o x i d a t i v e d e g r a d a t i o n t o l o s e C26/27 o f the s i d e  (Scheme  in  to i n s u f f i c i e n t m a t e r i a l .  Scheme 31 w i t h the a d d i t i o n o f methanol to the C-6  A  these  by the sponge or  An e t h a n o l e x t r a c t was  to i s o l a t e the e t h y l e s t e r d e r i v a t i v e .  i n c o n c l u s i v e due  118  4)  the  The  side  t o be the  first  118 Scheme 30:  -  B i o s y n t h e t i c p r o p o s a l f o r A-nor s t e r o i d s 118,  119  - 119 -  120  G.  The Bruker  -"-H NMR s p e c t r a WP-80,  were  EXPERIMENTAL  recorded  Nicolet-Oxford  on  either  270 o r V a r i a n  t h e Bruker-WH-400,  XL-300  spectrometers.  T e t r a m e t h y l s i l a n e (6 = 0) was employed as t h e i n t e r n a l s t a n d a r d NMR s p e c t r a and CDC1  (5 = 77.0 ppm) o r DMS0-d  3  b o t h as i n t e r n a l s t a n d a r d s as w e l l unless otherwise Low  as  f o r ^H  (6 - 39.5 ppm) were used  6  solvents  for  1 3  C  NMR  spectra  indicated.  resolution  and h i g h  r e s o l u t i o n e l e c t r o n impact mass s p e c t r a  were r e c o r d e d on K r a t o s  MS-59  Infrared  r e c o r d e d on a P e r k i n - E l m e r 1710 FT s p e c t r o m e t e r  spectra  were  and MS-50  spectrometers  respectively.  and UV a b s o r p t i o n s p e c t r a were measured on a Bausch and Lomb 2000  spectrometer.  Optical  rotations  model 141 p o l a r i m e t e r u s i n g a 10 p o i n t s were determined HPLC  was  cm  were  cell,  measured on P e r k i n - E l m e r  while  uncorrected  c a r r i e d out on e i t h e r a P e r k i n - E l m e r S e r i e s 2 i n s t r u m e n t  equipped  detection.  with  a  Waters  440 d u a l wavelength  Waters  model  reverse  The s o l v e n t s used f o r HPLC were BDH Ombisolve  F i s h e r HPLC grade and the water used was g l a s s - d i s t i l l e d .  s o l v e n t s used were a t l e a s t reagent grade u n l e s s o t h e r w i s e Silica  A l l other  indicated.  g e l types used were Merck s i l i c a g e l 60 PF-254 f o r p r e p a r a -  t i v e TLC, Merck s i l i c a g e l 60 230-400 mesh f o r f l a s h chromatography Merck  silica  501  d e t e c t o r f o r peak  The HPLC column used was the Whatman Magnum-9 0DS-3  phase p r e p a r a t i v e column. or  melting  on a F i s h e r - J o h n s m e l t i n g p o i n t a p p a r a t u s .  equipped w i t h a P e r k i n - E l m e r LC-55 UV d e t e c t o r o r a system  Spectronic  g e l 60  and  PF-254 w i t h CaSO^ 1/2H 0 f o r r a d i a l TLC. A l l R 2  f  121  values  were  c a l c u l a t e d o n a n a l y t i c a l TLC p l a t e s u s i n g M a c h e r e y - N a g e l S i l  G / U V 254 p r e c o a t e d  Preparation  sheets  of methylated  To a s t i r r e d in distilled  . 2 5 mm t h i c k .  4-methyl-2-nitrophenol  suspension  acetone  (7 m l )  4-methyl-2-nitrophenol  of potassium i n a 25 m l  (1.000  g,  the  this,  methyl  iodide  round  monitored  (1.800  b y TLC ( 1 0 : 9 0  g,  into  a  separatory  over  anhydrous  y e l l o w gum  Na2S04,  which  acetate/petroleum oil:  X  ether)  gave 3  m/z o b s e r v e d intensity)  (dd,  J -  167.0583,  167  (78),  9,  the  was  by  (5 m l ) . minutes.  ether).  were  while  filtrate  red was  (25 m l )  combined,  dried  E v a p o r a t i o n i n vacuo y i e l d e d a  3H), 3.95 7.60  TLC  (d,  (s,  (10:90  J -  (27),  a (d,  ethyl yellow J  -  2 H z , I H ) ppm; HRMS,  Q  105  as  3H), 6.99  for C H N03 167.0583; 120 ( 8 2 ) ,  and  The  between water  preparative  (s,  8  added  minutes  resulting  and p a r t i t i o n e d  2 Hz, IH),  (49),  40  compound 4 1 , 4 5 0 . 6 mg (41%)  5 2.35  required  137  and  filtered.  purification  H NMR ( 8 0 M H z , C D C 1 )  9 Hz, I H ) , 7.35  (75).  on  1 0 . 9 mmol)  1 2 . 7 mmol) was a d d e d d r o p w i s e  The c h l o r o f o r m l a y e r s  and  g,  flask  ethyl acetate/petroleum  funnel  a n d c h l o r o f o r m (4 x 30 m l ) .  bottom  approximately  r e a c t i o n m i x t u r e was v a c u u m f i l t e r e d poured  (1.500  completely red i n about 2  r e a c t i o n m i x t u r e was r e f l u x e d f o r  being  carbonate  6 . 5 mmol) d i s s o l v e d i n a c e t o n e  The r e s u l t i n g y e l l o w s o l u t i o n t u r n e d After  41  91  MS m / z (100),  (relative 77 ( 5 0 ) ,  65  122 Preparation of Brominated Compound 51  P u r i f i e d N-brominosuccinimide (0.155 g, .87 mmol) was refluxing  solution  of 41  (.124  tetrachloride (5 ml) i n a 10 ml  g,  round  .81 mmol) bottom  added  dissolved  flask.  The  to a  i n carbon refluxing  mixture was i r r a d i a t e d f o r 1 hr with a 150 W tungsten l i g h t bulb. cooling, the mixture was f i l t e r e d and the f i l t r a t e was vacuo. (3:10  The  After  concentrated i n  r e s u l t i n g yellow heavy o i l was p u r i f i e d by preparative TLC  ethyl acetate/hexanes) giving a clear yellow o i l (Rf. 51), 0.089 g  (45%):  MS,  m/z  (relative  intensity)  247 (2), 245 (3), 166 (65), 134  (51), 120 (25), 91 (34), 77 (62).  Preparation of methylated phidolopin 53  Purified  compound  tetrahydrofuran theophylline tion  was  51  (0.080  (3 ml) and added  g,  0.36  mmol)  was  dissolved  in  dropwise to a s t i r r i n g solution of  (0.051 g, 0.28 mmol) i n 0.1 M NaOH (2.5 ml).  After  complete, the reaction mixture was refluxed f o r 1 hr.  addiAt the  end of t h i s time the reaction mixture was cooled and partitioned between distilled  water  (15 ml) and chl oroform (4 x 20 ml).  The chloroform  layer was dried over anhydrous Na2S04 and f i l t e r e d .  Evaporation of the  solvent  TLC (ethyl acetate)  followed  by  purification  by  preparative  yielded 0.043 g (81%) of compound 53 as a white s o l i d :  1  H NMR (80 MHz,  CDC1 ) 3.43 (s, 3H), 3.58 (s, 3H), 5.48 (s, 2H), 7.00 (d, J - 9 Hz, IH), 3  7.38 (dd, J = 9, 2 Hz, IH), 7.68 (d, J -  2  Hz,  IH) ppm;  HRMS,  m/z  123 -  observed  345.1075, r e q u i r e d f o r C  1 5  H 5N 0 1  5  5  345.1075;  MS, m/z  (relative  i n t e n s i t y ) 345 (M, 7 ) , 180 (23), 166 (100), 91 (21), 77 ( 1 4 ) .  P r e p a r a t i o n o f the MOM p r o t e c t e d 4 - m e t h y l - 2 - n i t r o p h e n o l  To a s t i r r e d s u s p e n s i o n o f anhydrous potassium 6.5  mmol) i n d i s t i l l e d  acetone  42  carbonate  (5 m l ) .  room  temperature.  methylmethyl  19.5  mmol)  (1.50 ml,  r e s u l t a n t y e l l o w s o l u t i o n was s t i r r e d mixture funnel  was  10  extracted  ml  distilled  with  portions  y e l l o w c o l o r had d i s a p p e a r e d . anhydrous  Anhydrous  minutes.  The  turned chloro-  was added dropwise  f o r 10  6.5  and the reaction  f i l t e r e d and the f i l t r a t e was added t o a 150 ml s e p a r a t i v e  containing  repeatedly  (1.000 g,  The r e s u l t i n g y e l l o w s o l u t i o n  deep r e d a f t e r 15-20 minutes a t ether  g,  (20 ml) under a n i t r o g e n atmosphere i n a  50 mL round bottom f l a s k was added 4 - m e t h y l - 2 - n i t r o p h e n o l mmol) d i s s o l v e d i n acetone  (0.897  Na2S0^  and  water. of  The  aqueous  chloroform  layer  (25 ml) u n t i l the  The combined o r g a n i c l a y e r was d r i e d  filtered.  Evaporation  was  over  o f the s o l v e n t i n vacuo  f o l l o w e d by p r e p a r a t i v e TLC (1:10 e t h y l a c e t a t e / h e x a n e s ) y i e l d e d 0.930 (73%)  of  compound  42  as  a  yellow  oil:  *H  NMR  (80 MHz, C D C l ) 5 3  2.35-3.53 ( s , 3H), 5.25 ( s , 2H), 7.13 (bd, J - 9 Hz, I H ) , 7.30 (dd, J 9,  2  Hz, I H ) , 7.56 (d, J - 2 Hz, IH) ppm; HRMS, m/s observed  required f o r C H 9  167  1 1  N0  4  197.0688;  MS m/z ( r e l a t i v e  (34), 166 (2.5), 136 (11), 45 (100).  g  intensity)  =  197.0693, 197 ( 5 5 ) ,  124  Phidolopin 55  Preparation of MOM  Purified  N-brominosuccinimide  refluxing solution of tetrachloride  (5  42  ml)  (.0891  in  a  g,  10  g, 1.36 mmol) was added to a  (.242 .45  mmol)  ml round bottom flask.  mixture was i r r a d i a t e d for 15 minutes with a cooling, vacuo. ml)  the  added  reaction  dropwise  carbon  The refluxing  sun  lamp.  After  to  in  tetrahydrofuran  water  (5  a solution of theophylline (.0823 g, .045 After addition was  complete,  mixture was s t i r r e d at room temperature for 36 hours.  the end of this distilled  W  in  mixture was f i l t e r e d and the f i l t r a t e was concentrated i n  mmol) and .1 M sodium hydroxide (2.5 ml). the  250  The r e s u l t i n g brown s o l i d was dissolved  and  dissolved  time  the  reaction  (15  ml)  and  mixture  was  partitioned  chloroform (4 x 20 ml).  layer was dried over anhydrous Na S04 and f i l t e r e d . 2  At  between  The chloroform  Evaporation of  the  solvent followed by p u r i f i c a t i o n by preparative TLC (ethyl acetate) gave .534 g (32%) of pure 55 as a white s o l i d : H NMR l  (s,  3H),  3.50  (s, 3H), 3.71  (bd, J = 8.5 Hz, IH), 7.53 (d,  J  =  2.1  Hz,  16 17 5°6  375.1180;  (M  2),  C  H  +  N  + H 0,  (79).  2  330  IH)  (s, 3H), 5.25  MS (6),  3  (s, 2H), 5.46  (dd, J - 8.5, 2 Hz, IH), 7.63 ppm;  CDC1 ) 6  (80 MHz,  (s, 2H),  (s,  IH),  3.40 7.28  7.75  HRMS, m/z observed 375.1178, required for  m/z  (relative  intensity)  375  (8),  393  313  (21), 258 (27), 209 (32), 180 (100), 95  125 Preparation of Phidolopin  (36)  Deprotection of the MOM derivative was (.025  g,  .067 mmol) dissolved  achieved  by  refluxing  i n chloroform (3 ml) i n 50% acetic acid  plus one drop of concentrated sulphuric  acid for 1  hour.  the reaction was monitored v i a TLC (ethyl acetate).  Progress  (4 x  20  The chloroform layer was dried over sodium sulphate and f i l t e r e d .  Evaporation of the solvent ative  TLC  (ethyl  yellow needles: 3.41 7.63  of  The yellow reaction  mixture was partitioned between water (10 ml) and chloroform ml).  54  i n vacuo, followed by p u r i f i c a t i o n by prepar-  acetate)  gave .0215 g (97%) X  3  (s, IH) ppm;  (100), 9100):  7.65 (s, IH),  8.06 (d, J - 1.9 Hz, IH),  HRMS, m/z observed 331.0908, required  for C H N 0 1 4  +  (29),  123 (21), 95 (30); 3  124.94,  5  5  180  UV ( C H C I 3 ) 355 (e 1900), 275.8 (e  C NMR (750 MHz, CDCl -DMS0-d ) 5 27.62, 29.46, 47.99,  119.71,  1 3  EI-LRMS m/z ( r e l a t i v e intensity) 331 (M , 91), 313 (20),  152 1 3  6  (s, 3H), 5.48 (s, 2H), 7.16 (bd, H = 8.5 Hz, IH),  (dd, J - 1.9, 8.5 Hz, IH),  331.0917;  .19),  f  mp 212-213°C (CHCI3); H NMR (80 MHz, CDCl -DMS0-d ) 6  (s, 3H), 3.59  10.6  of Phidolopin (R  105.89,  6  127.88, 135.48, 142.21, 148.58, 151.11, 152.56, 154.57  ppm.  Preparation of MOM desmethylphidolopin 56  P u r i f i e d N-brominosuccinimide (1.470 g, 8.25 mmol) was added refluxing  solution  tetrachloride  of 42  (5 ml) i n a 10  (.41  g,  ml  round  2.1  mmol)  bottom  dissolved  flask.  The  to a  i n carbon refluxing  - 126 mixture  was  irradiated  for  90  minutes with a 250 W sun lamp.  cooling, the mixture was  f i l t e r e d and the f i l t r a t e was  vacuo.  brown s o l i d was  The  resulting  ml) and added dropwise to a solution mmol)  and 0.1  16 hours.  of  M NaOH (3.5 ml) which was  concentrated  in  dissolved i n tetrahydrofuran  (5  3-methylxanthine  (.3  solvent  dried over anhydrous Na2S04 and f i l t e r e d . followed  6  CDC1 ) 3  3^35  IH), 7.66 ppm;  (s, 3H),  m/z  1  observed  (s, 3H),  IH), 7.95  ( r e l a t i v e intensity) 361  166  152  (75),  (34), 134  (61), 121  (s, 2H),  (d, J - 5 Hz,  361.1025,  EI-LRMS m/z  5.44  chloro-  Evaporation of  of pure 56 as a white s o l i d : H NMR  3.44  (dd, J = 9, 2 Hz,  HRMS,  The  by p u r i f i c a t i o n by r a d i a l preparative  acetate) gave 0.026 g (25%)  1.8  partitioned  between d i s t i l l e d water (15 ml) and chloroform (4 x 25 ml).  the  g,  s t i r r e d at room temperature for  At the end of this time, the reaction mixture was  form layer was  After  7.38  IH),  TLC  (ethyl  (80  MHz,  (d, J - 9 Hz, 8.26  (s,  IH)  required for C ^ H ^ ^ O g 361.1495; (76), 343  (30), 105  (31), 329  (46), 299  (38),  (38), 94 (63), 77 (95),  51  (100).  Preparation  of D e s m e t h y l p h i d o l o p i n  Deprotection of the MOM (0.026  g, 0.071  (37)  derivative was  achieved  by  mmol) dissolved i n chloroform (3 ml)  was  mixture was ml).  The  monitored  via  TLC  (ethyl acetate).'  p a r t i t i o n e d between water (10 ml) and  chloroform layer was  55  i n 50% acetic acid  plus one drop of concentrated sulphuric acid for 1 hr. reaction  refluxing  Progress of  the  The yellow reaction  chloroform  dried over anhydrous Na2S0^ and  (4  x  20  filtered.  - 127 Evaporation  of  the  solvent i n vacuo, followed by p u r i f i c a t i o n by pre-  parative TLC (ethyl acetate) gave .024 g (37) (R  f  .15) as a yellow s o l i d :  X  (94%) of  desmethylphidolopin  H NMR (80 MHz, CDC1 ) 8 3.46 (s, 3H), 3  5.45 (s, 2H), 7.10 (d, J - 8.5 Hz, IH), 7.68 (dd, J - 8.5, 1.9 Hz, IH), 7.82  (s, IH), 8.05  observed (relative (45),  106  317.0760,  (37), 95 6  8  -  required  intensity)  CDCl -DMS0-d ) 3  (d, J  317  1.9 Hz, IH), 11.03 (s, IH); HRMS, m/z  for C H N 0  (M+,  (55), 77  1 3  1 1  5  5  317.0761;  EI-LRMS  m/z  2), 299 (10), 166 (100), 152 (49), 123 (53), 68  (80);  1 3  C  NMR  (75  MHz,  28.5, 47.9, 106.3, 119.9, 125.2, 127.6, 135.2, 142.3,  150.4, 151.1, 152.8, 154.9 ppm.  128  BRYOZOANS  Collection  Data  Bryozoans Barkley tion,  Sound, the  three  normally  depths  animals  from  were  they  were one  2 to  SCUBA  at  10 m.  Immediately  i n methanol  If  animals  s t o r e d at  the  lower  Diaperoecia  californica  (653  g d r i e d weight  with  the  a Waring blender animals.  through  celite,  filtrate  w h i c h was  between  brine  red  Filtration,  in  vacuo,  ml)  acetate  followed  red crude  oil.  collec-  for  one  to  immediately  (about  2 C)  until  after  extraction)  C  used,  yielding  by  to  (1  L)  used for  ground bryozoans  acetate  were  evaporation,  (3  dried  filtered methanolic  300  ml a n d  x 400  ml).  The  combined  over  anhydrous  Na2S0^.  i n vacuo,  Flash chromatographic  was  extraction  was  a r e d d i s h brown aqueous  approximately  and e t h y l layers  1892)  methanol  The s u s p e n s i o n o f  concentrated  (200  ethyl  not  in  Separation  (d'Ornigny  whole  after  and s t o r e d were  temperature  locations  month.  and C h r o m a t o g r a p h i c  in  various  immersed  californica  the  dark  at  using  Diaperoecia  ground  dark  collected  room t e m p e r a t u r e .  within  Extraction  of  B.C.  whole  days at  worked u p ,  1.  were  gave  5.8  fractionation  partitioned  g  (40  (.89%)  of  a  mm d i a m e t e r  - 129 column, 15 cm s i l i c a gel, step gradient 5% ethyl acetate/hexanes to 100% ethyl  acetate)  yielded fractions containing fats, s t e r o l s , pigments as  well as three strongly absorbing UV bands on TLC (100% ethyl Purification  of the most  non polar  band  Sephadex LH-20 (7:3 methanol/chloroform)  acetate).  by column chromatography,  yielded  a  strongly  retained  yellow band corresponding to 4-hydroxymethyl-2-nitrophenol (38) (4.5 mg, .007%, Rf. 35). Column chromatography of the next most using  Sephadex  LH-20  (7:3 methanol/chloroform)  polar  afforded  fraction a strongly  retained yellow band corresponding to phidolopin (36) (1.0 mg, <.001%, Rf.  24). P u r i f i c a t i o n of the most polar band i n a s i m i l a r fashion gave  desmethylphidolopin (37) (3.7 mg, <.001%, R . 15). f  Phidolopin (36): mp 211-212°C (CHC1 ); UV 275  (e 16800);  % NMR (270 MHz,  CDCI3)  A  (CH3CN)  3  8  m a x  351 nm (e 3300),  10.55 (s, IH, exchanges with  D 0), 8.09 (d, J - 2.5 Hz, IH), 7.63 (s, IH), 7.61 (dd, J = 2.5, 8.5 Hz, 2  IH), 7.17 (d, J - 8.5 Hz, IH), 5.46 (s, 2H), 3.59 (s, 3H), 3.40 (s, 3H); HRMS observed m/z 331.0914, required f o r C H 3 N 0 14  1  5  5  331.0917;  LRMS, m/z  ( r e l a t i v e i n t e n s i t y ) 331 (14), 313 (25), 180 (100), 152 (38).  Desmethylphidolopin  (37): ^-H NMR (80 MHz,  CDCI3)  5 10.60 (s, IH), 8.06  (d, J = 2 Hz), 7.63 (s, IH), 7.59 (dd, J = 2, 9 Hz), 7.13 (d, J - 9 Hz), 5.39  (s, 2H), 3.51 (s, 3H) ppm; HRMS observed m/z 317.0760, calculated  for C H N 0 1 3  1 1  5  5  317.0761;  LRMS, m/z ( r e l a t i v e i n t e n s i t y ) 317 (4),  299  - 130 (12), 166 (100), 152 (52), 123 (45), 106 (37), 95  (55), 77  (54),  68  (80), 51 (71).  4-hydroxymethyl-2-nitrophenol  (38): H NMR (80 MHz, CDCI3) 6 10.53 (s, 1  IH), 8.09 (d, J - 2 Hz), 7.58 (dd, J - 2, 9 Hz, IH), 4.69 (s, 2H) ppm;, HRMS m/z 169.0375, calculated for C H N0 7  7  4  169.0375;  LRMS, m/z ( r e l a t i v e  intensity) 169 (100), 152 (11), 123 (25), 122 (11), 106 (16), 95  (15),  77 (25), 65 (45).  4-methoxymethyl-2-nitrophenol  (39):  X  H NMR (80 MHz, CDCI3) 5 10.58 (s,  IH), 8.10 (d, J - 2 Hz, IH), 7.60 (dd, J - 2, 9 Hz, IH), 4.43 (s, 3.44  2H),  (s, 3H) ppm; HRMS observed m/z 183.0535, calculated f o r CgHqN04  183.0532;  LRMS, m/z ( r e l a t i v e intensity) 183 (59), 182 (29), 152 (100),  141 (25), 136 (21), 123 (12), 106 (39), 105 (14), 77 (24), 65 (12).  2.  The  remaining  bryozoans,  Heteropora alaskensis.  insculpta. T r i c e l l a r i a ternata. were extracted for IL_ c a l i f o r n i c a .  Hippodiplosia  as described  The results are shown i n the Table 13.  above  - 131  Table 13:  Nitrophenols  from Northeast P a c i f i c Bryozoans  *Crude EtOAc Extract  Organism  1  Diaperoecia  2  Heteropora alaskensis  5.7 (1.20)  3  Hippodiplosia insculpta  4  T r i c e l l a r i a ternata  g  californica  (%  Yield 36  37  mg 38  (%) 39  4.5 ( .007)  -  -  1.9 (<.001)  -  3.7 (1.50)  -  3.6 (<.001)  5.2 ( -72)  -  5.8 ( -89)  dry  1.0 ( .001)  weight  3.7 (<.001)  -  after  2.4 (<.001)  3.4 (<.001)  extraction)  - 132 ANTHOARCUATA GRACEAE (Bakus 1966)  C o l l e c t i o n Data  Anthoarcuata graceae was c o l l e c t e d during a l l locations  i n Barkley  seasons  Sound, B.C. at depths of 1 to 5 metres.  ately a f t e r c o l l e c t i o n , the sponge was immersed i n methanol and  at various  stored at room temperature f o r up to three days.  or  Immediethanol  I f the sponge was  not worked up immediately, i t was stored at low temperature  (4-(-5)"C)  u n t i l used ( t y p i c a l l y within 2 weeks).  Extraction and Chromatographic Separation  During  the course  of study on the extracts of the marine sponge  Anthoarcuata graceae. a number of c o l l e c t i o n s were made y i e l d i n g or  no  observed  variation  i n metabolites.  little  Therefore, the following  represents a t y p i c a l procedure. After storage at room temperature for 2 days, nolic  layer  was  decanted  and stored  the aqueous  metha-  at room temperature while the  sponge, approximately 1200 g (dry weight a f t e r extraction)  was  further  soaked i n dichloromethane (6 L) for 12 hours before being transferred i n dichloromethane into a Waring blender and ground This  crude  slurry  down  into  a  slurry.  was vacuum f i l t e r e d , along with the i n i t i a l aqueous  methanolic layer, through c e l i t e and the deep red f i l t r a t e  was  concen-  133 trated to about 250 ml before being partitioned between and  dichloromethane (4 x 30 ml).  were then dried over anhydrous filtration  were  brine  (15  ml)  The combined dichloromethane extracts  Na2S0 .  The  4  sponge  solids  from  the  transferred back into the o r i g i n a l c o l l e c t i o n jars and  soaked i n dichloromethane three more times at 1 day i n t e r v a l s .  The same  procedure of f i l t r a t i o n , p a r t i t i o n i n g of f i l t r a t e and drying the organic layers over anhydrous Na2S0 was repeated each day. 4  The combined dichloromethane extracts trated  in  vacuo  to  yield  sponge a f t e r extraction). raphy  (50  mm  a  were  filtered  and  crude dark red o i l , 46.9 g (3.9% of dry  The o i l was fractionated by f l a s h  chromatog-  diameter column, 16 cm s i l i c a gel, step gradient of 100%  hexanes to 30% ethyl acetate/hexanes) to y i e l d crude fractions ing  fats,  analytical  concen-  contain-  pigments,  carotenoids and a mixture of steroids detected by  TLC  ethyl  (50%  acetate/hexanes).  The  combined  steroid  containing fractions (charring a bright pink colour with 50% H2SO4 spray reagent) were pooled and evaporated to y i e l d 8.2 g crude  red  oil.  the  steroidal  c a r r i e d out using repeated column chromatography packed with 1 m of Sephadex LH-20, 55% to  The s t i l l  impure  further  of  a  still  Further p u r i f i c a t i o n of this complex mixture of fats,  carotenoids and pigments i n addition to  solvent)  (.68%)  components  was  (20 mm diameter column,  methanol/chloroform  as  running  y i e l d fractions containing fats, carotenoids and steroids.  purified  steroidal by  fraction  preparative  from  column  chromatography  was  reverse phase HPLC (10% water/aceto-  n i t r i l e ) to y i e l d two major peaks (retention times 40.6 min. min., flow rate of 2.8 ml/min., UV A  m a x  and  47.4  254 nm) which each were found to  possess 3 novel minor steroids as detected by a n a l y t i c a l  TLC  analysis.  134  Final  o f the t h r e e components from each HPLC  s e p a r a t i o n and p u r i f i c a t i o n  out by p r e p a r a t i v e T L C (5% methanol/  peak were c a r r i e d yielding  A  compound  .0006%,  R  .64),  f  (.0064  compound  .0005%, R f  g,  F (.0037  D (.0073 g ,  .74), compound  .0003%, R  g,  dichloromethane)  .42)  f  from  the  second  peak w i t h r e t e n t i o n time 47.4 min.  Compound cast) 3031, 0.73 (s,  116:  2956,  2871,  ( s , 3H), 0.95 5.09  3H),  CDCI3)  6  92-93°C  mp  1686,  (d, J -  (t,J -  6.4  1643,  6.0 Hz,  199.56,  202.36,  (CH3CN); UV 2 4 8 . 9 nm  Hz, IH),  161.06,  1606  -1  3H),  1.17  6.19  ( s , IH)  ppm;  125.48,  39.14,  35.98,  35.56,  35.50,  27.99,  25.74, 2 4 . 6 9 ,  23.98,  20.89,  18.55,  17.51,  (relative 257  (19),  required 396  intensity) 243  (18),  137  for  (26),  (52),  105  (19),  312  (20), 95  (CHCI3  1 3  NMR  C  124.99,  34.22, 11.91  81  MHz,  (75  56.55,  55.91,  34.17,  33.98,  EI-HRMS,  ppm;  EI-LRMS  (68), 283  (55),  1.69  ( s , 3H),  396.3030;  C27H40O2  381  1.61  ( s , 3H),  4 6 . 8 3 , 42.57,  396.3034,  IR  3  50.99,  observed  11600);  ^-H NMR (270 MHz, CDC1 ) 6  cm ;  127.09,  (e  (46),  (52),  m/z  270  69  m/z  (13),  (92),  55  (100).  Compound 12500);  X  1.02  4.73  (s, IH),  (d, J -  observed  21.99,  8.0  6.17  156.65,  39.10, 3 5 . 6 2 , 22.70,  111-112°C  mp  Hz,  3H),  ( s , IH) 125.44,  35.51,  34.50,  21.84,  410.3182,  ppm;  1.17 1 3  C  106.06,  34.18,  20.86,  required  ( s , 3H),  ( s , 3H),  (CH3CN)  0.85  3.49  (d,  J  2 4 8 . 6 nm (e -  4.65  ( s , 3H),  7.0  56.61,  55.77,  33.96,  30.89,  17.51,  C 8H4 0 2  2  2  29.70,  11.88 410.3187;  199.56,  46.79,  50.93,  27.98,  ppm;  Hz,  (s, IH),  NMR (75 MHz, CDCI3) 202.36,  18.62,  for  UV  (CH3CN):  H NMR (270 MHz, CDCI3) 6 0.73  3H),  161.08,  117:  42.55, 23.95,  EI-HRMS,  EI-LRMS,  m/z m/e  - 135 ( r e l a t i v e intensity) 410 (36), 327 (76), 297 (19), 283 (49), 257 (53), 137 (800, 109 (42), 95 (52), 81 (62), 69 (83), 55 (100).  Compound  118:  mp  142-143°C  (CH3CN); UV (CH3CN) A  m a x  250.2 nm (e  12500); IR (CHCI3 cast) 3552, 3021, 2951, 2869, 1744, 1717, 1 8  cm ;  64  1  H  (400 MHz, CDCI3) S 0.73 (s, 3H), 0.96 (d, J = 6.7 Hz, 3H), 1.21  NMR  (s, 3H), 1.62 (s, 3H), 1.62 (s, 3H), 1.69 (s, 3H), 1.88 (d,  -1  (m,  IH),  2.08  J = 13.3 Hz, IH), 2.19 (d, J = 13.3 Hz, IH), 2.41 (m, IH), 3.79 (s,  3H), 5.10 ( t , J = 8.0 Hz, IH), 6.73 ( t , J - 3.3 Hz, IH)  ppm;  1 3  C  NMR  (75 MHz, CDCI3) S 200.91, 173.37, 145.10, 136.28, 131.05, 125.09, 79.90, 56.26, 56.02, 55.91, 53.39, 49.66, 42.94, 39.92, 32.32, 12.03  32.21, ppm;  442.3079;  28.11,  EI-HRMS, EI-LRMS  39.35,  36.06,  35.55,  25.75, 24.74, 24.35, 22.11, 21.79, 18.66, 17.66, m/z  m/z  observed (relative  442.3083, intensity)  required  f o r C H 204 28  4  442 (79), 427 (24), 383  (10) , 358 (23), 340 (18), 329 (41), 311 (19), 298  (30), 283  (7), 269  (11) , 135 (32), 147 (22), 107 (41), 95 (58), 81 (49), 69 (100), 55 (68).  Compound 119:  Needles; mp (CH3CN) 155-157°C; UV (CH3CN) A  m a x  249.3  (e 12034); IR (CHCI3, cast), 3022, 2929, 2856, 1744, 1717, 1643 cm" ; 1  NMR  X  H  (400 MHz, CDCI3) S 0.73 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.04 (d,  J = 6.5 Hz, 3H), 1.20 (s, 3H), 1.89 (m, IH), 2.10 (d, J - 13.5 Hz, 2.18  (d, J  -  13.5 Hz, IH), 2.39 (m, IH), 3.77 (s, 3H), 4.65 (s, IH),  4.71 (s, IH), 6.73 ( t , J = 2.5 Hz, IH) ppm; 200.91,  173.36,  156.79,  145.08,  1 3  C NMR  30.99,  28.13,  24.34,  (75 MHz,  CDCI3) 6  136.29, 106.01, 79.89, 56.25, 55.93,  53.40, 49.64, 46.68, 42.95, 39.93, 39.34, 35.72, 32.21,  IH),  34.64,  33.81,  32.32,  22.11, 22.01, 21.87, 21.79, 18.74, 12.03  - 136 ppm;  EI-HRMS, m/z observed 456.3236, required  EI-LRMS 298  m/z  (relative  f o r C29H44O4 456.3241;  intensity) 456 (2), 396 (2), 367 (23), 353 (5),  (48), 284 (10), 269 (65), 147 (35), 124 (56), 109 (49), 95 (73),  81  (68), 69 (89), 55 (100).  Compound 120:  1  H NMR (400 MHz, CDCI3) 5 0.73 (s, 3H), 0.93 (d, J =  6 Hz, 3H), 1.28 (d, J = 8 Hz, 3H), 1.52 (s, 3H), 1.69 (s, 3H), 2.18 (d, J  -  16  Hz, IH), 2.25 (d, J = 16 Hz, IH), 3.29 (s, 3H), 4.52 ( t , J - 3  Hz, IH), 5.09 ( t , J - 7 Hz, IH), 5.28 (s, IH) ppm; EI-HRMS, m/z observed 414.3149,  required  f o r C27H42O3  414.3136;  EI-LRMS  m/z  intensity) 414 (19), 382 (28), 268 (10), 330 (16), 298 (15), 245  (relative 269 (29),  (30), 161 (26), 138 (34), 109 (34), 97 (52), 81 (49); 69 (100), 55  (81);  UV 264.7 nm (e 10971, c - .04) + NaOH (304.7 nm, e 9731).  Compound 121: H NMR (400 MHz, CDCI3) S 0.73 (s, 3H), 0.94 (d, J = X  8  Hz,  3H),  1.03  (d, J - 7 Hz, 6H), 1.31 (s, 3H), 2.17 (d, J = 16 Hz,  IH), 2.29 (d, J - 16 Hz, IH), 3.33 (s, 3H), 4.57 ( t , J - 2 Hz, IH), 4.69 (s,  IH), 4.76 (s, IH), 5.28 (s, IH) ppm; EI-HRMS, m/z observed 428.3284  required (5),  f o r C28H40O3 428.3291; EI-LRMS m/z  396  (13), 344  (relative  intensity)  428  (12), 312 (14), 300 (12), 271 (24), 161 (30), 138  (37), 107 (38), 95 (52), 81 (61), 69 (95), 55 (100).  - 137 Preparation of 4-ene-3,6-dione 122 by Jones oxidation  The Jones reagent was (26.72  g,  prepared  by  dissolving  chromium  trioxide  270 mmol) i n 50 ml d i s t i l l e d water i n a 100 ml beaker.  beaker was immersed i n an ice water bath and concentrated s u l f u r i c 911.5  ml,  200 mmol)  was  acid  added followed by enough d i s t i l l e d water to  bring the t o t a l volume fo 100 ml. 2.6  The  A solution of cholesterol  (1.00 g,  mmol dissolved i n 30 ml acetone was s t i r r e d i n a 50 ml round bottom  f l a s k at 0°C i n an ice water bath before 2 ml of the previously prepared cold  oxidation  reagent  was  added  at a  mixture temperature of around 20°C. hours,  with  the reaction  rate to maintain a reaction  The s t i r r i n g was maintained  for 3  mixture turning yellowish from the o r i g i n a l  deep red colour.  After 3 hours, 10 ml of methanol was added to destroy  excess  and the reaction mixture was f i l t e r e d through c e l i t e .  reagent,  The r e s u l t i n g l i g h t brown solution was placed into a 150 ml separatory funnel and extracted with chloroform (4 x 50 ml). further washed with 50 ml of brine and dried sulfate.  Filtration,  followed  The organic layer was  over  anhydrous  by evaporation i n vacuo y i e l d s a l i g h t  brown gum (462 mg) which on preparative TLC yuielded one highly ing  UV  magnesium  absorb-  band at Rf .50 (30% ethyl acetate/hexanes) which based on spec-  t r a l data corresponded to the ene-dione 122, 337.9 mg (32.7%).  Compound 122: 1685,  mp 132-134°C (CH CN); IR (CHC1 3  3  cast)  1606 cm' ; H NMR (270 MHz, CDCI3) S 0.74 (s, 3H), 0.87 (d, J = 6 1  X  Hz, 6H), 0.94 (d, J = 6 Hz, 3H), 1.19 (s, 3H), 6.19 (s, NMR  2953, 2871,  (75 MHz,  CDCI3)  5  IH) ppm;  1 3  C  202.25, 199.40, 160.98, 125.33, 56.42, 55.82,  - 138 50.83, 46.71, 39.70, 39.36, 39.01, 35.57, 35.40, 23.86,  23.69,  22.75,  20.77,  18.55,  observed 398.3186 required f o r C21 M°2 Y[  intensity)  392  17.40,  34.09, 11.79  398.3187;  33.87,  27.91,  ppm; EI-HRMS, m/z  EI-LRMS m/z  (relative  (2), 383 (14), 370 (6), 343 (2), 329 (6), 243 (40), 147  (24), 135 (33), 124 (35), 105 (37), 91 (65), 79 (64), 67 (55), 55 (100).  Anthosterone B-acetate (123)  2.8 mg (.006 mmol) of Compound D 119 was s t i r r e d acetic  anhydride  room temperature.  (2:1)  i n pyridine  and  i n a 10 ml round bottom f l a s k f o r 12 hours at  Evaporation of the solvent and  excess  reagent  gave  acetylated product 123, as a white s o l i d , i n quantitative y i e l d .  Compound 123:  W NMR  l  (270 MHz, CDCI3) 6 0.72 (s, 3H), 0.97 (d, J =  8 Hz, 3H), 1.03 (d, J - 8 Hz, 6H), 1.10 (s, 3H), 2.16 (s, 3H), 2.47 J  =  14  Hz,  (d,  IH), 2.65 (d, J = 15 Hz, IH), 3.76 (s, 3H), 4.67 (s, IH),  4.73 (s, IH), 6.81 ( t , J = 4 Hz, IH) ppm; EI-HRMS, m/z observed 498.3347 required  f o r C3;iH4 05 6  498.3347;  EI-LRMS m/z ( r e l a t i v e i n t e n s i t y ) 498  (1), 483 (2), 456 (37), 382 (4), 312 (28), 287 (27), 135 (28), 123 (27), 109 (36), 95 (57), 81 (57), 69 (93), 55 (100).  - 139 APPENDIX 1  Bioassay r e s u l t s f o r phidolopin (36)  Phidolopin significant  (36)  levels  and  and desmethylphidolopin  (37)  desmethylphidolopin  vitro  antifungal  activity  against Pythiam ultimum (75 and  demonstrated  of a c t i v i t y i n the standard disk minimum i n h i b i t o r y  concentration bioassays performed i n our laboratory. in  (37)  Rhizoctonia solani  with a minimum i n h i b i t o r y  ug/.25  (70  Phidolopin  i n disk),  showed  concentration  Helminthosporium satium  ug/.25 i n disk) and a n t i b a c t e r i a l a c t i v i t y  against B a c i l l u s s u b t i l i s 19 ug/.25 i n disk)  and  Staphlococcus aureus  (75 ug/.25 i n disk). 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